Dietary peptides

ABSTRACT

The present invention relates to novel peptides, composition comprising such peptides including nutritional supplements and methods for inducing satiation and satiety, for weight management and preventing or reducing the incidence of obesity, or for preventing or reducing cardiovascular diseases, atherosclerosis, hypertension, hepatosteatosis, cancer and/or diabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofPCT/EP2016/081572 filed Dec. 16, 2016, which International Applicationwas published by the International Bureau in English on Jun. 22, 2017,and claims priority from European Application No. 15200440.4, filed Dec.16, 2015, which applications are hereby incorporated by reference intheir entirety in this application.

FIELD OF THE INVENTION

The present invention relates to novel peptides, composition comprisingsuch peptides including nutritional supplements and methods for inducingsatiation and satiety, for preventing or reducing the incidence ofmetabolic syndrome comprising overweight and obesity, cardiovasculardiseases, atherosclerosis, hypertension, hepatosteatosis, diabetesand/or cancer.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 20549US00_July_2021_ST25.txt, a creation date of Jul. 23, 2021,and a size of 191 Kb. The sequence listing filed via EFS-Web is part ofthe specification and is hereby incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

Obesity is a common medical condition affecting numerous humansthroughout the world and is associated with, induces or increases therisk of developing conditions such as cardiovascular diseases,atherosclerosis, hypertension, hepatosteatosis, cancer and/or diabetes.

Some regulators of obesity have been identified. However, despiteintensive study, the regulation of obesity is still poorly understood.

Protein is more satiating than carbohydrate and fat, and its effect onfood intake is more than can be accounted for by its energy contentalone. The mechanism by which proteins trigger food intake regulatorysystems is unclear. However, it seems likely that satiety signalsarising from protein ingestion begin in the gastrointestinal tract uponproteolytic digestion.

Accordingly, dietary proteolytic products (peptides and amino acids)induce signalling in enteroendocrine cells of the intestine, which leadsto secretion of various gut hormones, e.g. glucagon-like peptide-1(GLP-1) (FIG. 1) with neuronal, local (auto- and paracrine) and systemic(endocrine) effects (FIG. 2), ultimately leading to satiation (amount offood ingested as a meal) and satiety (length of time between meals). Itis well-known that (some) enteroendocrine cells respond to free aminoacids and small peptides (di- and tripeptides), which are readily takenup by the enterocytes and metabolized and/or transported into systemiccirculation. Rate of digestion, i.e. transit time in the GI tract,secretion of digestive enzymes, etc, is a highly regulated process,where cellular responses to undigested proteins and/or increases inamino acids and peptides in the gut leads to secretion of gut hormones,e.g. GLP-1, peptide tyrosine-tyrosine (PYY), neurotensin (NT), whichinduces satiation. If these signals persist in the gut because of slowand prolonged release, satiety is enhanced. One such mechanism is theileal brake, where unknown components in partly digested food reachesthe distal small intestine and invokes a response in the form ofsecretion of the gut hormones GLP-1, PYY, NT and possibly others, as yetunknown hormones. However, the precise mechanism behind the ileal brakeis unknown.

The specific peptide(s) responsible for this satiety inducing signal(s)is largely unknown and it would be of great importance if any of thesepeptides could be identified.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide newpolypeptides that induce or signals satiety in a subject.

The polypeptides of the invention may be used to treat conditionsassociated with a wide variety of metabolic diseases, for use in weightmanagement, and/or for preventing or reducing the incidence ofoverweight and/or obesity, or for preventing or reducing cardiovasculardiseases, atherosclerosis, hypertension, hepatosteatosis, cancer and/ordiabetes.

SUMMARY OF THE INVENTION

Dietary proteolytic products (peptides and amino acids) inducesignalling in enteroendocrine cells of the intestine, which leads tosecretion of various gut hormones, e.g. GLP-1 (FIG. 1) with both central(CNS), local (auto- and paracrine) and systemic (endocrine) effects(FIG. 2), ultimately leading to satiation and satiety.

It has been found by the present inventor(s) that novel meat-derivedpolypeptides are superior in signalling of intestinal cell lines (FIG.3) and that only very specific peptides are capable of signalling (FIG.4). The inventors of the present invention have identified polypeptidesincluding an octapeptide (ASDKPYIL, SEQ ID NO:6) present in proteolyticdigests (FIG. 5) and resistant to pepsin degradation, of which apentapeptide (KPYIL, SEQ ID NO:9) is the minimal sequence withsignificant biologic activity (FIG. 6). The octapeptide sequence isunique for the muscle-specific alpha-actinin-2 protein, and the sequenceis conserved between all animal species. This peptide would beapplicable as a novel, but natural nutritional supplement to inducesatiation and satiety.

So, in a first aspect the present invention relates to an isolatedpolypeptide comprising the amino acid sequenceAA1-AA2-AA3-K-AA5-AA6-AA7-AA8  (formula I, SEQ ID NO:1),wherein AA1 is an optional amino acid selected from A, L, I, and V; AA2is an optional amino acid selected from S, T, G, A, N, E and D; AA3 isan optional amino acid selected from D, E, and G; AA5 is selected fromP, N, S, D, A, T, K, and G; AA6 is selected from Y, N, I, W, and F; AA7is selected from I, L, R, and V; AA8 is selected from L, I, V, S, M, andT; which polypeptide is not more than 50 amino acids in length; or avariant thereof with a sequence identity of at least 80%.

In a second aspect the present invention relates to an isolatedpolypeptide consisting of the amino acid sequenceR1-AA1-AA2-AA3-K-AA5-AA6-AA7-AA8-R2  (formula II, SEQ ID NO:2),wherein AA1 is an optional amino acid selected from A, L, I, and V; AA2is an optional amino acid selected from S, T, G, A, N, E and D; AA3 isan optional amino acid selected from D, E, and G; AA5 is selected fromP, N, S, D, A, T, K, and G; AA6 is selected from Y, N, I, W, and F; AA7is selected from I, L, R, and V; AA8 is selected from L, I, V, S, M, andT; R1 defines the N-term (—NH2) or a protection group; R2 defines theC-term (—COOH).

In a third aspect the present invention relates to a polypeptide havingor comprising a sequence selected from ASDKPYIL (SEQ ID NO:6), SDKPYIL(SEQ ID NO:7), DKPYIL (SEQ ID NO:8), KPYIL (SEQ ID NO:9), AGDKNYIL (SEQID NO:10), AGDKNYIT (SEQ ID NO:11), AGDKSYIT (SEQ ID NO:12), ADGKPYIV(SEQ ID NO:13), AEDKDFIT (SEQ ID NO:14), AADKPYIL (SEQ ID NO:15),ATDKPYIL (SEQ ID NO:16), AGDKPYIT (SEQ ID NO:17), ASEKPYIL (SEQ IDNO:18), ADGKPYVT (SEQ ID NO:19), AGDKPYIL (SEQ ID NO:20), ASDKPNIL (SEQID NO:21), ASDKPYIT (SEQ ID NO:22), AADKPFIL (SEQ ID NO:23), ASDKAYIT(SEQ ID NO:24), AGDKAYIT (SEQ ID NO:25), ANGKPFIT (SEQ ID NO:26),AGDKNFIT (SEQ ID NO:27), ASDKSYIT (SEQ ID NO:28), ASDKTYIT (SEQ IDNO:29), ASDKNYIT (SEQ ID NO:30), AGDKKYIT (SEQ ID NO:31), AGDKNYIS (SEQID NO:32), AADKNYIT (SEQ ID NO:33), AGDKNYIM (SEQ ID NO:34), AADKNFIM(SEQ ID NO:35), AADKNFIT (SEQ ID NO:36), and AGDKGIRS (SEQ ID NO:37).

In a fourth aspect the present invention relates to a compositioncomprising a polypeptide of the invention.

In a further aspect the present invention relates to a polypeptideaccording to the invention for use in promoting satiety in a subject,for use in weight management, and/or for preventing or reducing theincidence of overweight and/or obesity in a subject, or for preventingor reducing cardiovascular diseases, atherosclerosis, hypertension,hepatosteatosis, cancer and/or diabetes.

In a further aspect the present invention relates to a method ofpreventing or reducing the incidence of obesity in a subject, and/or ofpromoting satiety in a subject, and/or to reduce or treat cardiovasculardiseases, atherosclerosis, hypertension, hepatosteatosis, cancer and/ordiabetes comprising enteral administering to a subject in need thereof apolypeptide comprising or consisting of the amino acid sequenceAA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8  (formula III, SEQ ID NO:3),wherein AA1 is an optional amino acid selected from A, L, I, and V; AA2is an optional amino acid selected from S, T, G, A, N, E and D; AA3 isan optional amino acid selected from D, R, K, E, and G; AA4 is an aminoacid selected from K and R; AA5 is selected from P, N, S, D, A, T, K,and G; AA6 is selected from Y, N, I, W, and F; AA7 is selected from I,L, R, and V; AA8 is selected from L, I, V, S, M, and T; whichpolypeptide is not more than 50 amino acids in length; or a variantthereof with a sequence identity of at least 80%.

In a further aspect the present invention relates to a compositionaccording to the invention for use in promoting satiety in a subject,and/or for use in weight management, and/or for preventing or reducingthe incidence of obesity in a subject and/or for use in preventing orreducing cardiovascular diseases, atherosclerosis, hypertension,cardiovascular diseases, high blood pressure, cancer and/or diabetes.

In a further aspect the present invention relates to a method ofpromoting satiety in a subject, and/or of preventing or reducing theincidence of obesity in a subject, and/or to reduce or treatcardiovascular diseases, atherosclerosis, hypertension, cardiovasculardiseases, high blood pressure, cancer and/or diabetes, comprisingadministering to a subject in need thereof a composition according tothe invention.

LEGENDS TO THE FIGURES

FIG. 1. Dose-response curve for effect of protein hydrolysate on releaseof GLP-1 from GLUTag cells (open circles) or a control cell line (closedsymbols) that does not produce GLP-1. Cells (˜5×10^5 per sample) wereincubated for up to 90 min in Dulbeccos Modified Eagle Medium (DMEM)containing 5.56 mM glucose in absence or presence of different amounts(weight/volume) of meat protein hydrolysate. Supernatant was filteredthrough 0.45 micron filters and assayed for content of GLP-1 asdescribed in ELISA protocol. Data are mean+SEM from quadruplicatesamples.

FIG. 2. Signaling by dietary nutrients in enteroendocrine cells.Illustration from Horm Res Paediatr. 2015; 83(1):1-10.

FIG. 3. Stimulation of cell signaling (measured as increase inintracellular fluorescence) by meat protein hydrolysates (filledsymbols) or whey protein hydrolysates (open symbols) in three differentintestinal cell lines: Top) a murine intestinal cell line; middle)GLUTag cells; bottom) CaCo2 cells.

FIG. 4. Size exclusion fractionation of protein hydrolysate and test ofbiologic activity. Absorbance at 280 nm shown by thick, solid line,activity of fractions by filled circles.

FIG. 5. Verification of identified sequence ASDKPYIL by syntheticpeptide. Comparison of dose-response relationship of meat hydrolysateand pure, synthetic peptide identified by sequencing of purifiedfractions.

FIG. 6. Identification of minimal active sequence in ASDKPYIL in murine(mIC) and human (hIC) intestinal cells.

Truncation from the amino-terminal or from the carboxy-terminal end ofASDKPYIL has different consequences. Deleting the carboxy-terminalleucine reduces potency more than two orders of magnitude in mIC cellsand abrogates activity in hIC. Peptides with further deletions of 2, 3or 4 amino acids from the carboxy-terminus are without activity.Deleting the first three amino acids from the amino-terminus has no bigimpact on activity. However, the fourth amino acid, lysine, is critical,since PYIL has two orders of magnitude lower activity compared with thefull sequence in mIC and no activity in hIC. The following peptides arerepresented in FIG. 6: PYIL (SEQ ID NO: 4), KPYIL (SEQ ID NO: 9), DKPYIL(SEQ ID NO: 8), SDKPYIL (SEQ ID NO: 7), ASDKPYIL (SEQ ID NO: 6), ASDKPYI(residues 1-7 of SEQ ID NO: 6), ASDKPYI (residues 1-6 of SEQ ID NO: 6),ASDKPY (residues 1-5 of SEQ ID NO: 6). ASDKP (residues 1-5 of SEQ ID NO:6), ASDK (residues 1-4 of SEQ ID NO: 6).

FIG. 7. Identification of critical residues in ASDKPYIL (SEQ ID NO: 6)(d-Ala (A_(D)) scan). Systematic replacement of all residues in ASDKPYIL(SEQ ID NO: 6) with the d-isomer of alanine and corresponding biologicalactivity. Results show that 1) the last four amino acids (PYIL, SEQ IDNO:4) are critical, 2) replacing K reduces potency more than 30-fold, 3)replacing the aspartic residue improves potency almost 10-fold, and 4)alanine and serine on the first two positions are without importance.

FIG. 8. Stability of peptides in rodent intestine. 0.001 mg/ml of theindicated peptides were incubated with pieces of rodent intestine (mouseand rat intestine gave similar results) for up to 10 minutes at 37 oC.Recovery of activity was tested with dose-response curves as indicated.The following peptides are represented in FIG. 8: RRPYIL (SEQ ID NO:39), DKPYIL (SEQ ID NO: 8), ASDKPYIL (SEQ ID NO: 6), AADKPYIL (SEQ IDNO: 15),

FIG. 9. Stability of peptides in rodent intestine. EC50 values fordifferent peptides and different incubation times were calculated fromFIG. 8 and recovered activity plotted as a function of time. Thefollowing peptides are represented in FIG. 9: ASDKPYIL (SEQ ID NO: 6),RRPYIL (SEQ ID NO: 39), DKPYIL (SEQ ID NO: 8). AADKPYIL (SEQ ID NO: 15).

FIG. 10. Comparison of the sequences of three known gut hormones,neurotensin, neuromedin N (SEQ ID NO: 1010) and xenin (SEQ ID NO: 1011)with that of DC7-2 (ASDKPYIL, SEQ ID NO: 6). The PYIL (SEQ ID NO: 4)sequence is conserved, although Y is replaced by W in xenin.

FIG. 11. Comparison of the DC7-2 sequence (aa 891-898) in isoforms ofa-actinin 2 (Hs:Homo sapiens ACTN1 (SEQ ID NO: 1012); ACTN2 (SEQ ID NO:1013); ACTN3 (SEQ ID NO: 1014); ACTN4 (SEQ ID NO: 1015)) andconservation between species (Dm: Drosophila melanogaster (DmACTN, SEQID NO: 1016); Ce: Caenorhabditis elegans (CeACTN, SEQ ID NO: 1017); Dd:Dictyostelium discoideum (DdACTN, SEQ ID NO: 1018); Sp:Schizosaccharomyces pombe (SpACTN, SEQ ID NO: 1019); Dr: Danio rerio).The following peptides are represented in FIG. 11: AGDKNYIT (SEQ ID NO:11), AGDKNFIT (SEQ ID NO: 27), ASDKPYIL (SEQ ID NO: 6). AADKPYIL (SEQ IDNO: 15). ASGKTFIT(SEQ ID NO: 1006), AEDKDFIT (SEQ ID NO: 14). ADGKSYVT(SEQ ID NO: 1007).

FIG. 12. 24 Balb/c female mice, 10-11 weeks, 20-22 g, were acclimatizedto 12 h dark light cycle and placed single-housed in metabolic cages.Following administration of the indicated doses of DC7-2, feed and waterintake was monitored for 6 h.

FIG. 13. Summary of cell signaling activities of N-terminalsubstitutions in octa-, hepta-, hexa- and pentapeptides based on thesequence of DC7-2. Single-letter abbreviations for the 20 amino acidsare shown on the plot centered at the corresponding EC50. The nativeamino acid in DC7-2 is marked with a grey circle for each of thepeptides. The following peptides are represented in FIG. 13: SDKPYIL(SEQ ID NO: 7), DKPYIL (SEQ ID NO: 8), KPYIL (SEQ ID NO: 9), PYIL (SEQID NO: 4).

FIG. 14. Stability of DC7-2 families of peptides in intestinehomogenates. Single-letter abbreviations for the 20 amino acids areshown on the plot with the corresponding stability expressed as thelogarithm to the concentration of intestine homogenate that degradeshalf of the activity of peptide. All peptides were incubated at 10-5 Mwith various dilutions of a homogenate of the entire small intestine(pool from 20 mice). After incubation for 90 min at 37 oC, degradationwas stopped by addition of 1 M phosphoric acid (final 0,4 M, pH ˜1.2).Each peptide incubation mix was neutralized with NaOH and immediatelytested for activity in intestinal cells. Control for zero degradation,i.e. addition of phosphoric acid before addition of intestinehomogenate, was included for each peptide. The native amino acid inDC7-2 is marked with a grey circle for each of the peptides. Thefollowing peptides are represented in FIG. 14: SDKPYIL (SEQ ID NO: 7),DKPYIL (SEQ ID NO: 8), KPYIL (SEQ ID NO: 9), PYIL (SEQ ID NO: 4).

FIG. 15. Stability of DC7-2 families of peptides in serum. The followingpeptides are represented in FIG. 15: SDKPYIL (SEQ ID NO: 7). DKPYIL (SEQID NO: 8), KPYIL (SEQ ID NO: 9), PYIL (SEQ ID NO: 4).

FIG. 16. Stability of X-KPYIL hexapeptides in intestine homogenate andserum. The following peptides are represented in FIG. 16: KPYIL (SEQ IDNO: 9).

FIG. 17. 24 Balb/c female mice, 10-11 weeks, 20-22 g, were acclimatizedto 12 h dark light cycle. Mice were divided into four groups each of sixmice and placed single-housed in metabolic cages. Mice were thenadministered vehicle alone (day 1) for monitoring of feed and waterintake for 6 h. On day 3, the same groups received the indicated dosesof DC7-2, and feed and water intake was monitored for 6 h.

FIG. 18. Swiss Webster male mice, 25-30 g, were acclimatized to 12 hdark/light cycle and placed single-housed in cages. Followingadministration just prior to onset of dark cycle of vehicle alone (0.5ml of PBS w 1% of BSA) or vehicle+DC7-2, feed intake was monitored everyhour for 6 h (during dark cycle). Mean and SEM from four experiments,each with 6-8 mice per treatment. Data were fitted with linearregression (R2>0.99) and 95% confidence intervals are shown as greylines. Accumulated feed intake for treatment with DC7-2 was 64%+/−5%compared with control for these four experiments.

FIG. 19. Swiss Webster male mice, 25-30 g, were acclimatized to 12 hdark/light cycle and placed single-housed in cages. Followingadministration just prior to onset of dark cycle of vehicle alone (0.5ml of PBS w 1% of BSA) or vehicle+DC7-2, feed intake was monitored everyhour for 12 h (during dark cycle) and then intermittently up to 30 h.

FIG. 20. Swiss Webster male (25-30 g) or female (20-25 g) mice wereacclimatized to 12 h dark/light cycle and placed in groups of 6-8 miceper cage. Vehicle (0.5 ml of PBS w 1% of BSA) alone or vehicle+DC7-2 wasadministered three times per day (08:00; 16:00; 24:00), and feed intakewas monitored daily for a week. Data were fitted with linear regression(R2>0.99) and 95% confidence intervals (grey lines).

DETAILED DISCLOSURE OF THE INVENTION

The inventors of the present invention have found novel polypeptidesthat may be used to induce signalling in intestinal cells and mayconsequently induce satiety. Although a specific peptide has beenidentified from a proteolytic digest of muscle-specific alpha-actinin-2protein, it is envisioned that similar polypeptides will bind the samereceptors in the intestine and provide the same biological activity,i.e. signal to induce satiation and satiety. Similar peptides maycontain e.g. conservative substitutions or be truncated. The rationalefor using the polypeptides of the invention is that the energy contentdue to the relatively small length of the peptide is low as compared tothe effect on satiety.

Definitions

When terms such as “one”, “a” or “an” are used in this disclosure theymean “at least one”, or “one or more” unless otherwise indicated.Further, the term “comprising” is intended to mean “including” and thusallows for the presence of other constituents, features, conditions, orsteps than those explicitly recited.

In some specific embodiments, the first 1, 2, or 3 amino acids in theN-terminal of the amino acid sequences according to the invention are inthe D-form. It is assumed that the N-terminal trimming and therebydegradation of the peptides are somewhat delayed by having amino acidsof the D-form in the N-terminal of these polypeptides. Alternatively andin some embodiments, the first 1, 2, or 3 amino acids in the N-terminalof the amino acid sequences according to the invention are amino acidsin beta or gamma forms. Beta amino acids have their amino group bondedto the beta carbon rather than the alpha carbon as in the 20 standardnatural amino acids. A capital D-letter subscript after the letterrepresenting the amino acid residue designate herein amino acidsspecified to be in D-form, such as W_(D) referring to a tryptophan inD-form. A capital L-letter subscript after the letter representing theamino acid residue designate herein amino acids specified to be inL-form, such as W_(L) referring to a tryptophan in L-form. If nototherwise indicated, an amino acid is in its natural L-form.

Alternatively, the first 1, 2, or 3 amino acids in the N-terminal of theamino acid sequences according to the invention may be modified byincorporation of protective groups, e.g. fluorine, or alternativelycyclic amino acids or other suitable non-natural amino acids are used.

A “variant” or “analogue” of a peptide refers to a peptide having anamino acid sequence that is substantially identical to a referencepeptide, typically a native or “parent” polypeptide, or a polypeptide offormula I or II. The peptide variant may possess one or more amino acidsubstitutions, deletions, and/or insertions at certain positions withinthe native amino acid sequence. The “variant” within this definitionstill has functional activity. In some embodiment a variant has at least80% sequence identity with the reference polypeptide. In someembodiments a variant has at least 85% sequence identity with thereference polypeptide. In other embodiments a variant has at least 90%sequence identity with the reference polypeptide. In a furtherembodiment a variant has at least 95% sequence identity with thereference polypeptide.

“Conservative” amino acid substitutions are those in which an amino acidresidue is replaced with an amino acid residue having a side chain withsimilar physicochemical properties. Families of amino acid residueshaving similar side chains are known in the art, and include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Aparticular form of conservative amino acid substitutions include thosewith amino acids, which are not among the normal 20 amino acids encodedby the genetic code. Since preferred embodiments of the presentinvention entail use of synthetic peptides, it is unproblematic toprovide such “non-naturally occurring” amino acid residues in thepeptides disclosed herein, and thereby it is possible to exchange thenatural saturated carbon chains in the side chains of amino acidresidues with shorter or longer saturated carbon chains—for instance,lysine may be substituted with an amino acid having a side chain—(CH2)nNH3, where n is different from 4, and arginine may be substitutedwith an amino acid having the side chain (CH2)nNHC(═NH2)NH2, where n isdifferent from 3, etc. Similarly, the acidic amino acids aspartic acidand glutamic acid may be substituted with amino acid residues having theside chains —(CH2)nCOOH, where n>2.

The polypeptides of this invention may in some embodiments benefit fromhaving higher stability than polypeptides containing only naturallyoccurring amino acids, and its modification enables to have much higherstability, such as a modification in the N-terminal of the polypeptide.

Accordingly and in some embodiments, the polypeptides of this inventionhave at their N-terminal a protection group, such as a protection groupselected from the group consisting of acetyl group, fluorenyl methoxycarbonyl group, formyl group, palmitoyl group, myristyl group, stearylgroup and polyethylene glycol (PEG).

The active peptide may also be di- or multimerized, e.g. throughcross-linking with suitable di- or multivalent chemical cross-linkers,e.g. disuccinimidyl suberate, containing spacers of different length,e.g. 10-100 Å, and different functionality, e.g. homo- orheterofunctional, for coupling through non-critical amino or otherreactive groups. Alternatively, photoactivation or enzymaticcross-linking may be used to increase stability and potency in vivo.

The modifications of peptides described above greatly increase thestability of the peptides of this invention. The term used herein“stability” refers to in vivo stability, such as the stability in thegut of a subject receiving such polypeptide. The protection groupdescribed above protects the peptides from the attack of protease invivo.

The polypeptides according to the invention may be derived from aproteolytic digests of meat and be resistant to pepsin degradation.Accordingly, in some embodiments a polypeptide according to theinvention may only contain naturally occurring amino acids.

In other embodiments, a polypeptide according to the invention is morestable towards degradation in the gastrointestinal tract, e.g. asmeasured in a stability assay described in the examples of the presentinvention, as compared to a control peptide. In some embodiments, apolypeptide according to the invention is more stable towardsdegradation in the gastrointestinal tract, e.g. measured in a stabilityassay described in the examples of the present invention as compared toa control peptide with the sequence RRPYIL, (SEQ ID NO:39).

In some embodiments, a polypeptide according to the invention has anhalf-life (T½) of degradation in vivo in the gut or in vitro, e.g.measured in a stability assay described in the example 2 of the presentinvention, which is higher than 2 min, such as higher than 4 min, suchas higher than 6 min, such as higher than 8 min, such as higher than 10min, such as higher than 15 min, such as higher than 20 min, such ashigher than 25 min, such as higher than 30 min, such as higher than 35min, such as higher than 40 min, such as higher than 45 min, such ashigher than 50 min, such as higher than 55 min, such as higher than 60min.

The term “substantially identical” in the context of two amino acidsequences means that the sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at leastabout 50, at least about 60, at least about 70, at least about 80, atleast about 90, at least about 95, at least about 98, or at least about99 percent sequence identity. In some embodiments, when measuring thesequence identity between two different peptide sequences, a gap of oneor two amino acids is allowed when the two peptide sequences are alignedwithout having any influence on the value of sequence identity. In someembodiments, a residue position that is not identical differ by only aconservative amino acid substitution. Sequence identity is typicallymeasured using sequence analysis software. Protein analysis softwarematches similar sequences using measures of similarity assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, the publiclyavailable GCG software contains programs such as “Gap” and “BestFit”which can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences can also be compared using FASTA or ClustalW,applying default or recommended parameters. A program in GCG Version6.1., FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson, Methods Enzymol. 1990; 183:63-98;Pearson, Methods Mol. Biol. 2000; 132:185-219). Another preferredalgorithm when comparing a sequence to a database containing a largenumber of sequences from various organisms is the computer programBLAST, especially blastp, using default parameters. See, e.g., Altschulet al., J. Mol. Biol. 1990; 215:403-410; Altschul et al., Nucleic AcidsRes. 1997; 25:3389-402 (1997); each herein incorporated by reference.“Corresponding” amino acid positions in two substantially identicalamino acid sequences are those aligned by any of the protein analysissoftware mentioned herein, typically using default parameters.

The term “functional activity” as used herein refers to a polypeptidethat stimulates cell signalling measured as fluorescence by elevatedintracellular calcium or cellular release of gut hormones, such asmeasured in the signalling assays described in the examples. Thefunctional activity of a variant may exhibit at least about 25%, such asat least about 50%, such as at least about 75%, such as at least about90% of the specific activity of a reference polypeptide, such as theoctapeptide ASDKPYIL, when tested in the assays as described herein.Alternatively, the functional activity of a variant may exhibit higheractivity than a reference polypeptide, such as the octapeptide ASDKPYIL,when tested in the assays as described herein.

An “isolated” molecule is a molecule that is the predominant species inthe composition wherein it is found with respect to the class ofmolecules to which it belongs (i.e., it makes up at least about 5% ofthe type of molecule in the composition and typically will make up atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or more of the species of molecules, e.g., peptides, in thecomposition). Commonly, a composition of a specific peptide sequence mayexhibit 90%-99% homogeneity for peptides in the context of all presentpeptide species in the composition or at least with respect tosubstantially active peptide species in the context of proposed use. Ifproduced synthetically, a composition of a specific peptide sequencewill exhibit 98%-99%, or even higher and close to 100% homogeneity forpeptides in the context of all present peptide species in thecomposition or at least with respect to substantially active peptidespecies in the context of proposed use.

Unless otherwise indicated the polypeptides within the present inventionis a linear sequence of amino acids. The term “linear sequence” as usedherein refers to the specific sequence of amino acids connected bystandard peptide bonds in standard N- to C-terminal direction. Thepeptide may contain only peptide bonds. In some embodiments however, asecond part of a peptide sequence may be bound to and continue from theside chain of a terminal amino acid in a first part of an amino acidsequence. Also the term does not exclude that an amino acid within asequence, such as within AA1-AA8, may be connected, such as through theside chains, with another amino acid at a distant location within thepeptide sequence, such as a distant location within AA1-AA8.

In the context of the present invention, “treatment” or “treating”refers to preventing, alleviating, managing, curing or reducing one ormore symptoms or clinically relevant manifestations of a disease ordisorder, unless contradicted by context. For example, “treatment” of apatient in whom no symptoms or clinically relevant manifestations of adisease or disorder have been identified is preventive or prophylactictherapy, whereas “treatment” of a patient in whom symptoms or clinicallyrelevant manifestations of a disease or disorder have been identifiedgenerally does not constitute preventive or prophylactic therapy.

The terms “patient” and “subject” refer to any human or animal that maybe treated using the methods of the present invention.

Many aspect of the present invention relates to the use of polypeptidesor compositions to promote satiety in a subject. The underlying cause ofa metabolic syndrome or disorder that may treated by the polypeptides orcompositions according to the invention, is an overconsumption ofcalories, while still not feeling satiety. By inducing or promotingsatiety in a subject, such total amounts of calories, including caloriesderived from fat and carbohydrates are reduced in the subject.Accordingly, the polypeptides and compositions of the invention may beused in preventing or reducing a metabolic syndrome or disorder, such asobesity, insulin-deficiency or insulin-resistance related disorders,Diabetes Mellitus (such as, for example, Type 2 Diabetes), glucoseintolerance, abnormal lipid metabolism, atherosclerosis, hypertension,cardiac pathology, stroke, non-alcoholic fatty liver disease,hyperglycemia, hepatic steatosis, dyslipidemia, dysfunction of theimmune system associated with overweight and obesity, cardiovasculardiseases, high cholesterol, elevated triglycerides, asthma, sleepapnoea, osteoarthritis, neuro-degeneration, gallbladder disease,syndrome X, inflammatory and immune disorders, atherogenic dyslipidemiaand cancer.

Preparation of Polypeptides of the Invention

The invention also relates to a method of preparing polypeptides of theinvention as mentioned above. The method of synthesis or preparationthereof includes, but is not limited to recombinant (whether producedfrom cDNA, genomic DNA, synthetic DNA or other form of nucleic acid),synthetic, and transgenic means.

The polypeptides of the invention described herein may be produced bymeans of recombinant nucleic acid techniques. In general, a nucleic acidsequence encoding the desired polypeptide is then inserted into anexpression vector, which is in turn transformed or transfected into hostcells.

As an alternative and also the preferred option, the polypeptides of theinvention are produced by synthetic means, i.e. by polypeptidesynthesis. In some embodiments, the invention relates to a method ofmanufacturing an analogue comprising non-natural amino acids from about5 total residues to about 20 total residues. In some embodiments, ananalogue comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 non-natural amino acids, such as any one of thefollowing non-naturally occurring amino acid residues.

The polypeptides of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, beta-alanine,desaminohistidine, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcys-teine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, nor-valine, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into polypeptides. For example, an invitro system can be employed wherein nonsense mutations are suppressedusing chemically aminoacylated suppressor tRNAs. Methods forsynthesizing amino acids and aminoacylating tRNA are known in the art.Transcription and translation of plasmids containing nonsense mutationsis carried out in a cell-free system comprising an E. coli S30 extractand commercially available enzymes and other reagents. Polypeptides arepurified by chromatography. See, for example, Robertson et al., J. Am.Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301,1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc.Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translationis carried out in Xenopus oo-cytes by microinjection of mutated mRNA andchemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol.Chem. 271:19991-8, 1996). Within a third method, E. coli cells arecul-tured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the polypeptide in place ofits natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

As another alternative to synthetic preparation, the polypeptides of theinvention may be purified from any natural source containing suchpolypeptide, such as from the proteolytic hydrolysate of muscle tissue,such as any source containing alpha-actinin-2 protein, such as by themethods described in the example section.

Accordingly, in some embodiments the sequence of the polypeptides of theinvention is derived from a sequence found in nature, such as a fragmentof alpha-actinin-2 protein.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing (IEF), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989). They may be purified by affinity chromatography on anantibody column. Additional purification may be achieved by conventionalchemical purification means, such as high performance liquidchromatography. Other methods of purification, including barium citrateprecipitation, are known in the art, and may be applied to thepurification—see, for example, Scopes, R., Protein Purification,Springer-Verlag, N.Y., 1982.

For the methods of the invention including the therapeutic purposes itis not critical to have a high purity of a specific peptide of theinvention. However, the higher the concentration of a specific peptideof the invention the higher is the effect in terms of inducing satiationand satiety relative to amount of total protein and total amount ofcalories consumed by the subject receiving the composition ofpolypeptides. It is to be understood that the idea of the invention isto administer polypeptides that induce satiation or satiety withoutadministering a lot of calories to the subject.

In some embodiments the compositions of polypeptides of the inventionare substantially pure. Thus, in an embodiment of the invention thepolypeptides of the invention are purified to at least about 90 to 95%homogeneity, preferably to at least about 98% homogeneity.

Purity may be assessed by e.g. HPLC and amino-terminal amino acidsequencing.

Administration and Pharmaceutical Compositions

Administration of the polypeptides according to the invention may bethrough several routes of administration, for example, lingual,sublingual, buccal, in the mouth, oral, in the stomach and intestine,nasal, pulmonary, for example, through the bronchioles and alveoli or acombination thereof, epidermal, dermal, transdermal, vaginal, rectal,ocular, for examples through the conjunctiva, uretal, and parenteral topatients in need of such a treatment.

Some kind of oral administration is preferred since these types ofpolypeptides are derived from a source that naturally has to passthrough the mouth and to the intestinal mucosa.

Compositions of the current invention may be administered in severaldosage forms, for example, as solutions, suspensions, emulsions,microemulsions, multiple emulsion, foams, salves, pastes, plasters,ointments, tablets, coated tablets, rinses, capsules, for example, hardgelatine capsules and soft gelatine capsules, suppositories, rectalcapsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops,ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginalrings, vaginal ointments, injection solution, in situ transformingsolutions, for example in situ gelling, in situ setting, in situprecipitating, in situ crystallization, infusion solution, and implants.

One of skill in the art will recognize that the appropriate dosage ofthe compositions and pharmaceutical compositions may vary depending onthe individual being treated and the purpose. For example, the age, bodyweight, and medical history of the individual patient may affect thetherapeutic efficacy of the therapy. Further, a lower dosage of thecomposition may be needed to produce a transient cessation of symptoms,while a larger dose may be needed to produce a complete cessation ofsymptoms associated with the disease, disorder, or indication. Acompetent physician can consider these factors and adjust the dosingregimen to ensure the dose is achieving the desired therapeutic outcomewithout undue experimentation. It is also noted that the clinicianand/or treating physician will know how and when to interrupt, adjust,and/or terminate therapy in conjunction with individual patientresponse. Dosages may also depend on the strength of the particularpolypeptide of the invention chosen for the pharmaceutical composition.

The dose of the composition or pharmaceutical compositions may vary. Thedose of the composition may be once per day. In some embodiments,multiple doses may be administered to the subject per day. In someembodiments, the total dosage is administered in at least twoapplication periods, In some embodiments, the period can be an hour, aday, a month, a year, a week, or a two-week period. In an additionalembodiment of the invention, the total dosage is administered in two ormore separate application periods, or separate doses.

In some embodiments, subjects can be administered the composition inwhich the composition is provided in a daily dose range of about 0.0001mg/kg to about 5000 mg/kg of the weight of the subject. The doseadministered to the subject can also be measured in terms of totalamount of polypeptide of the invention administered per day. In someembodiments, a subject is administered from about 0.001 to about 3000milligrams of polypeptide of the invention per day. In some embodimentsa subject is administered up to about 2000 milligrams of polypeptide ofthe invention per day. In some embodiments, a subject is administered upto about 1800 milligrams of polypeptide of the invention per day. Insome embodiments, a subject is administered up to about 1600 milligramsof polypeptide of the invention per day. In some embodiments, a subjectis administered up to about 1400 milligrams of polypeptide of theinvention per day. In some embodiments, a subject is administered up toabout 1200 milligrams of polypeptide of the invention per day. In someembodiments, a subject is administered up to about 1000 milligrams ofpolypeptide of the invention per day. In some embodiments, a subject isadministered up to about 800 milligrams of polypeptide of the inventionper day. In some embodiments, a subject is administered from about 0.001milligrams to about 700 milligrams of polypeptide of the invention perdose. In some embodiments, a subject is administered up to about 700milligrams of polypeptide of the invention per dose. In someembodiments, a subject is administered up to about 600 milligrams ofpolypeptide of the invention per dose. In some embodiments, a subject isadministered up to about 500 milligrams of polypeptide of the inventionper dose. In some embodiments, a subject is administered up to about 400milligrams of polypeptide of the invention per dose. In someembodiments, a subject is administered up to about 300 milligrams ofpolypeptide of the invention per dose. In some embodiments, a subject isadministered up to about 200 milligrams of polypeptide of the inventionper dose. In some embodiments, a subject is administered up to about 100milligrams of polypeptide of the invention per dose. In someembodiments, a subject is administered up to about 50 milligrams ofpolypeptide of the invention per dose.

A composition, wherein a polypeptide of the invention is added may beany food composition, food product, or food ingredient. Here, the term“food” is used in a broad sense—and covers food for humans as well asfood for animals (i.e. a feed). In a preferred aspect, the food is forhuman consumption. The food may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

When used as—or in the preparation of—a food—such as functional food—thecomposition of the present invention may be used in conjunction with oneor more of: a nutritionally acceptable carrier, a nutritionallyacceptable diluent, a nutritionally acceptable excipient, anutritionally acceptable adjuvant, a nutritionally active ingredient.

The composition of the present invention may be used as a foodingredient.

As used herein the term “food ingredient” includes a formulation whichis or can be added to functional foods or foodstuffs as a nutritionalsupplement. The term food ingredient as used here also refers toformulations which can be used at low levels in a wide variety ofproducts that require gelling, texturising, stabilising, suspending,film-forming and structuring, retention of juiciness and improvedmouthfeel, without adding viscosity.

The food ingredient may be in the form of a solution or as asolid—depending on the use and/or the mode of application and/or themode of administration.

The composition of the present invention may be—or may be added to—foodsupplements.

The composition of the present invention may be—or may be addedto—functional foods.

As used herein, the term “functional food” means food which is capableof providing not only a nutritional effect and/or a taste satisfaction,but is also capable of delivering a further beneficial effect toconsumer.

Accordingly, functional foods are ordinary foods that have components oringredients (such as those described herein) incorporated into them thatimpart to the food a specific functional—e.g. medical or physiologicalbenefit—other than a purely nutritional effect.

Although there is no legal definition of a functional food, most of theparties with an interest in this area agree that they are foods marketedas having specific health effects.

Some functional foods are nutraceuticals. Here, the term “nutraceutical”means a food which is capable of providing not only a nutritional effectand/or a taste satisfaction, but is also capable of delivering atherapeutic (or other beneficial) effect to the consumer. Nutraceuticalscross the traditional dividing lines between foods and medicine.

Surveys have suggested that consumers place the most emphasis onfunctional food claims relating to heart disease. Preventing cancer isanother aspect of nutrition which interests consumers a great deal, butinterestingly this is the area that consumers feel they can exert leastcontrol over. In fact, according to the World Health Organization, atleast 35% of cancer cases are diet-related. Furthermore claims relatingto osteoporosis, gut health and obesity effects are also key factorsthat are likely to incite functional food purchase and drive marketdevelopment.

The composition of the present invention can be used in the preparationof or added to food products such as one or more of: jams, marmalades,jellies, dairy products (such as milk or cheese), meat products, poultryproducts, fish products, vegetable-based soups, and bakery products.

By way of example, the composition of the present invention can be usedas ingredients to soft drinks, a fruit juice or a beverage comprisingwhey protein, health teas, cocoa drinks, milk drinks and lactic acidbacteria drinks, yoghurt and drinking yoghurt, cheese, ice cream, waterices and desserts, confectionery, biscuits cakes and cake mixes, snackfoods, breakfast cereals, instant noodles and cup noodles, instant soupsand cup soups, balanced foods and drinks, sweeteners, texture improvedsnack bars, fibre bars, bake stable fruit fillings, care glaze,chocolate bakery filling, cheese cake flavoured filling, fruit flavouredcake filling, cake and doughnut icing, heat stable bakery filling,instant bakery filling creams, filing for cookies, ready-to-use bakeryfilling, reduced calorie filling, adult nutritional beverage, acidifiedsoy/juice beverage, aseptic/retorted chocolate drink, bar mixes,beverage powders, calcium fortified soy/plaim and chocolate milk,calcium fortified coffee beverage.

A composition according to the present invention can further be used asan ingredient in food products such as American cheese sauce,anti-caking agent for grated & shredded cheese, chip dip, cream cheese,dry blended whip topping fat free sour cream, freeze/thaw dairy whippingcream, freeze/thaw stable whipped topping, low fat & lite naturalcheddar cheese, low fat Swiss style yoghurt, aerated frozen desserts,and novelty bars, hard pack ice cream, label friendly, improvedeconomics & indulgence of hard pack ice cream, low fat ice cream: softserve, barbecue sauce, cheese dip sauce, cottage cheese dressing, drymix Alfredo sauce, mix cheese sauce, dry mix tomato sauce and others.

For certain aspects, preferably the foodstuff is a beverage.

For certain aspects, preferably the foodstuff is a bakery product—suchas bread, Danish pastry, biscuits or cookies.

The present invention also provides a method of preparing a food or afood ingredient, the method comprising mixing a polypeptide according tothe present invention or the composition according to the presentinvention with another food ingredient.

Specific Embodiments of the Invention

One aspect of the invention related to an isolated polypeptidecomprising the amino acid sequenceAA1-AA2-AA3-K-AA5-AA6-AA7-AA8  (formula I, SEQ ID NO:1),wherein AA1 is an optional amino acid selected from A, L, I, and V; AA2is an optional amino acid selected from S, T, G, A, N, E and D; AA3 isan optional amino acid selected from D, E, and G; AA5 is selected fromP, N, S, D, A, T, K, and G; AA6 is selected from Y, N, I, W, and F; AA7is selected from I, L, R, and V; AA8 is selected from L, I, V, S, M, andT; which polypeptide is not more than 50 amino acids in length; or avariant thereof with a sequence identity of at least 80%.

Another aspect of the invention related to a method of promoting satietyin a subject or to a method of preventing or reducing the incidence ofobesity in a subject comprising enteral administering to a subject inneed thereof a polypeptide comprising or consisting of the amino acidsequenceAA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8  (formula III, SEQ ID NO:3),wherein AA1 is an optional amino acid selected from A, L, I, and V; AA2is an optional amino acid selected from S, T, G, A, N, E and D; AA3 isan optional amino acid selected from D, R, K, E, and G; AA4 is an aminoacid selected from K and R; AA5 is selected from P, N, S, D, A, T, K,and G; AA6 is selected from Y, N, I, W, and F; AA7 is selected from I,L, R, and V; AA8 is selected from L, I, V, S, M, and T; whichpolypeptide is not more than 50 amino acids in length; or a variantthereof with a sequence identity of at least 80%.

In the following AA1-AA8 may refer to the amino acids of either formulaI, II, or III.

In some embodiments AA1 is absent. In some embodiments AA1 is any onenatural amino acid selected from Y, W, V, T, S, R, Q, P, N, M, L, K, I,H, G, F, E, D, C, and A. In some embodiments AA2 is absent. In someembodiments AA2 is any one natural amino acid selected from Y, W, V, T,S, R, Q, P, N, M, L, K, I, H, G, F, E, D, C, and A. In some embodimentsAA3 is absent. In some embodiments AA1 is present. In some embodimentsAA2 is present. In some embodiments AA3 is present. In some embodimentsAA1 is A. In some embodiments AA2 is S. In some embodiments AA3 is D. Insome embodiments AA3 is selected from any one amino acid C, D, E, N, P,and Q. In some embodiments AA3 is selected from E and G. In someembodiments AA3 is P. In some embodiments AA3 is C. In some embodimentsAA4 is K. In some embodiments AA6 is Y. In some embodiments AA7 is I. Insome embodiments AA8 is L. In some embodiments the amino acid sequenceis not found in nature. In some embodiments AA8 is the C-terminal aminoacid. In some embodiments AA5 is P. In some embodiments AA6 is selectedfrom Y and W. In some embodiments AA7 is selected from I and L.

In some embodiments AA2 is an optional amino acid selected from S, T, A,N, E and D. In some embodiments AA5 is selected from P, S, D, A, T, K,and G. In some embodiments AA6 is selected from Y, N, I, and W. In someembodiments AA8 is selected from L, I, V, S, and M.

In some embodiments the polypeptide does not comprise any one of thesequences AVTEKKYILYDFSVTS (SEQ ID NO:5), PRRPYIL (SEQ ID NO:38), RRPYIL(SEQ ID NO:39), RPYIL (SEQ ID NO:40), RRPWIL (SEQ ID NO:41), KRPYIL (SEQID NO:42), KKPYIL (SEQ ID NO:43), Adamantoyl-KPYIL (SEQ ID NO:9),H-Lys-psi(CH₂NH)Lys-Pro-Tyr-Ile-Leu-OH (SEQ ID NO:44). In someembodiments the polypeptide does not comprise derivatives of Lys.

In some embodiments the polypeptide does not consists of any one of thesequences AVTEKKYILYDFSVTS (SEQ ID NO:5), PRRPYIL (SEQ ID NO:38), RRPYIL(SEQ ID NO:39), RPYIL (SEQ ID NO:40), RRPWIL (SEQ ID NO:41), KRPYIL (SEQID NO:42), KKPYIL (SEQ ID NO:43), Adamantoyl-KPYIL(SEQ ID NO:9),H-Lys-psi(CH₂NH) Lys-Pro-Tyr-Ile-Leu-OH (SEQ ID NO:44). In someembodiments the polypeptide is not a derivative of KPYIL (SEQ ID NO:9).

In some embodiments the amino acid sequence only contains natural aminoacids.

In some embodiments the polypeptide of the invention is 5-50, such as5-50, 5-49, 5-48, 5-47, 5-46, 5-45, 5-44, 5-43, 5-42, 5-41, 5-40, 5-39,5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27,5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, such as 5-18, such as5-17, such as 5-16, such as 5-15, such as 5-14, such as 5-13, such as5-12, such as 5-11, such as 5-10, such as 5-9, such as 5-8, such as 5-7,such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

In some embodiments the polypeptide of the invention is less than 50,49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32,31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, such as 18, such as17, such as 16, such as 15, such as 14, such as 13, such as 12, such as11, such as 10, such as 9, such as 8, such as 7 amino acids in length.

In some embodiments the polypeptide of the invention is 5-50, such as6-50, such as 7-50, such as 8-50, such as 9-50, such as 10-50, such as11-50, such as 12-50, such as 13-50, such as 14-50, such as 15-50, suchas 16-50, such as 17-50, such as 18-50, such as 19-50, such as 20-50,such as 21-50, such as 22-50, such as 23-50, such as 24-50, such as25-50, such as 26-50, such as 27-50, such as 28-50, such as 29-50, suchas 30-50, such as 31-50, such as 32-50, such as 33-50, such as 34-50,such as 35-50, such as 36-50, such as 37-50, such as 38-50, such as39-50, such as 40-50, such as 41-50, such as 42-50, such as 43-50, suchas 44-50, such as 45-50, such as 46-50, such as 47-50, such as 48-50,such as 49-50 amino acids in length.

In some embodiments the polypeptide of the invention is more than 5,such as 6, such as 7, such as 8, such as 9, such as 10, such as 11, suchas 12, such as 13, such as 14, such as 15, such as 16, such as 17, suchas 18, such as 19, such as 20, such as 21, such as 22, such as 23, suchas 24, such as 25, such as 26, such as 27, such as 28, such as 29, suchas 30, such as 31, such as 32, such as 33, such as 34, such as 35, suchas 36, such as 37, such as 38, such as 39, such as 40, such as 41, suchas 42, such as 43, such as 44, such as 45, such as 46, such as 47, suchas 48, such as more than 49 amino acids in length.

In some embodiments the polypeptide of the invention is an octapeptideor a heptapeptide

In some embodiments the polypeptide of the invention has or comprises asequence selected from ASDKPYIL (SEQ ID NO:6), SDKPYIL (SEQ ID NO:7),DKPYIL (SEQ ID NO:8), and KPYIL (SEQ ID NO:9).

In some embodiments the polypeptide of the invention consist of orcomprises a sequence selected from ASDKPYIL (SEQ ID NO:6), AGDKNYIL (SEQID NO:10), AGDKNYIT (SEQ ID NO:11), AGDKSYIT (SEQ ID NO:12), ADGKPYIV(SEQ ID NO:13), AEDKDFIT (SEQ ID NO:14), AADKPYIL (SEQ ID NO:15),ATDKPYIL (SEQ ID NO:16), AGDKPYIT (SEQ ID NO:17), ASEKPYIL (SEQ IDNO:18), ADGKPYVT (SEQ ID NO:19), AGDKPYIL (SEQ ID NO:20), ASDKPNIL (SEQID NO:21), ASDKPYIT (SEQ ID NO:22), AADKPFIL (SEQ ID NO:23), ASDKAYIT(SEQ ID NO:24), AGDKAYIT (SEQ ID NO:25), ANGKPFIT (SEQ ID NO:26),AGDKNFIT (SEQ ID NO:27), ASDKSYIT (SEQ ID NO:28), ASDKTYIT (SEQ IDNO:29), ASDKNYIT (SEQ ID NO:30), AGDKKYIT (SEQ ID NO:31), AGDKNYIS (SEQID NO:32), AADKNYIT (SEQ ID NO:33), AGDKNYIM (SEQ ID NO:34), AADKNFIM(SEQ ID NO:35), AADKNFIT (SEQ ID NO:36), and AGDKGIRS (SEQ ID NO:37).

In some embodiments the polypeptide of the invention is an isolatedpolypeptide.

In some embodiments the polypeptide of the invention is syntheticallymade.

In some embodiments the polypeptide of the invention is a purifiedfragment.

In some embodiments the polypeptide of the invention is purified fromanimal sources.

In some embodiments the polypeptide of the invention is generated byenzymatic treatment of proteins from animal sources.

In some embodiments the polypeptide of the invention has been modifiedby N terminal acylation or other chemical modifications to introduceprotection groups.

In some specific embodiments, the polypeptide of the invention consistsof or comprises an amino acid sequence selected from the groupconsisting of KPYIL, KPYII (SEQ ID NO:45), KPYIV (SEQ ID NO:46), KPYLL(SEQ ID NO:47), KPYLI (SEQ ID NO:48), KPYLV (SEQ ID NO:49), KPYVL (SEQID NO:50), KPYVI (SEQ ID NO:51), KPYVV (SEQ ID NO:52), KPWIL (SEQ IDNO:53), KPWII (SEQ ID NO:54), KPWIV (SEQ ID NO:55), KPWLL (SEQ IDNO:56), KPWLI (SEQ ID NO:57), KPWLV (SEQ ID NO:58), KPWVL (SEQ IDNO:59), KPWVI (SEQ ID NO:60), KPWVV (SEQ ID NO:61), RPYIL (SEQ IDNO:40), RPYII (SEQ ID NO:62), RPYIV (SEQ ID NO:63), RPYLL (SEQ IDNO:64), RPYLI (SEQ ID NO:65), RPYLV (SEQ ID NO:66), RPYVL (SEQ IDNO:67), RPYVI (SEQ ID NO:68), RPYVV (SEQ ID NO:69), RPWIL (SEQ IDNO:70), RPWII (SEQ ID NO:71), RPWIV (SEQ ID NO:72), RPWLL (SEQ IDNO:73), RPWLI (SEQ ID NO:74), RPWLV (SEQ ID NO:75), RPWVL (SEQ IDNO:76), RPWVI (SEQ ID NO:77), and RPWVV (SEQ ID NO:78).

In some specific embodiments, the polypeptide of the invention consistof or comprises an amino acid sequence selected from the groupconsisting of DKPYIL (SEQ ID NO:8), DKPYII (SEQ ID NO:79), DKPYIV (SEQID NO:80), DKPYLL (SEQ ID NO:81), DKPYLI (SEQ ID NO:82), DKPYLV (SEQ IDNO:83), DKPYVL (SEQ ID NO:84), DKPYVI (SEQ ID NO:85), DKPYVV (SEQ IDNO:86), DKPWIL (SEQ ID NO:87), DKPWII (SEQ ID NO:88), DKPWIV (SEQ IDNO:89), DKPWLL (SEQ ID NO:90), DKPWLI (SEQ ID NO:91), DKPWLV (SEQ IDNO:92), DKPWVL (SEQ ID NO:93), DKPWVI (SEQ ID NO:94), DKPWVV (SEQ IDNO:95), DRPYIL (SEQ ID NO:96), DRPYII (SEQ ID NO:97), DRPYIV (SEQ IDNO:98), DRPYLL (SEQ ID NO:99), DRPYLI (SEQ ID NO:100), DRPYLV (SEQ IDNO:101), DRPYVL (SEQ ID NO:102), DRPYVI (SEQ ID NO:103), DRPYVV (SEQ IDNO:104), DRPWIL (SEQ ID NO:105), DRPWII (SEQ ID NO:106), DRPWIV (SEQ IDNO:107), DRPWLL (SEQ ID NO:108), DRPWLI (SEQ ID NO:109), DRPWLV (SEQ IDNO:110), DRPWVL (SEQ ID NO:111), DRPWVI (SEQ ID NO:112), DRPWVV (SEQ IDNO:113), EKPYIL (SEQ ID NO:114), EKPYII (SEQ ID NO:115), EKPYIV (SEQ IDNO:116), EKPYLL (SEQ ID NO:117), EKPYLI (SEQ ID NO:118), EKPYLV (SEQ IDNO:119), EKPYVL (SEQ ID NO:120), EKPYVI (SEQ ID NO:121), EKPYVV (SEQ IDNO:122), EKPWIL (SEQ ID NO:123), EKPWII (SEQ ID NO:124), EKPWIV (SEQ IDNO:125), EKPWLL (SEQ ID NO:126), EKPWLI (SEQ ID NO:127), EKPWLV (SEQ IDNO:128), EKPWVL (SEQ ID NO:129), EKPWVI (SEQ ID NO:130), EKPWVV (SEQ IDNO:131), ERPYIL (SEQ ID NO:132), ERPYII (SEQ ID NO:133), ERPYIV (SEQ IDNO:134), ERPYLL (SEQ ID NO:135), ERPYLI (SEQ ID NO:136), ERPYLV (SEQ IDNO:137), ERPYVL (SEQ ID NO:138), ERPYVI (SEQ ID NO:139), ERPYVV (SEQ IDNO:140), ERPWIL (SEQ ID NO:141), ERPWII (SEQ ID NO:142), ERPWIV (SEQ IDNO:143), ERPWLL (SEQ ID NO:144), ERPWLI (SEQ ID NO:145), ERPWLV (SEQ IDNO:146), ERPWVL (SEQ ID NO:147), ERPWVI (SEQ ID NO:148), ERPWVV (SEQ IDNO:149), RKPYIL (SEQ ID NO:150), RKPYII (SEQ ID NO:151), RKPYIV (SEQ IDNO:152), RKPYLL (SEQ ID NO:153), RKPYLI (SEQ ID NO:154), RKPYLV (SEQ IDNO:155), RKPYVL (SEQ ID NO:156), RKPYVI (SEQ ID NO:157), RKPYVV (SEQ IDNO:158), RKPWIL (SEQ ID NO:159), RKPWII (SEQ ID NO:160), RKPWIV (SEQ IDNO:161), RKPWLL (SEQ ID NO:162), RKPWLI (SEQ ID NO:163), RKPWLV (SEQ IDNO:164), RKPWVL (SEQ ID NO:165), RKPWVI (SEQ ID NO:166), RKPWVV (SEQ IDNO:167), RRPYIL (SEQ ID NO:39), RRPYII (SEQ ID NO:168), RRPYIV (SEQ IDNO:169), RRPYLL (SEQ ID NO:170), RRPYLI (SEQ ID NO:171), RRPYLV (SEQ IDNO:172), RRPYVL (SEQ ID NO:173), RRPYVI (SEQ ID NO:174), RRPYVV (SEQ IDNO:175), RRPWIL (SEQ ID NO:41), RRPWII (SEQ ID NO:176), RRPWIV (SEQ IDNO:177), RRPWLL (SEQ ID NO:178), RRPWLI (SEQ ID NO:179), RRPWLV (SEQ IDNO:180), RRPWVL (SEQ ID NO:181), RRPWVI (SEQ ID NO:182), RRPWVV (SEQ IDNO:183), GKPYIL (SEQ ID NO:184), GKPYII (SEQ ID NO:185), GKPYIV (SEQ IDNO:186), GKPYLL (SEQ ID NO:187), GKPYLI (SEQ ID NO:188), GKPYLV (SEQ IDNO:189), GKPYVL (SEQ ID NO:190), GKPYVI (SEQ ID NO:191), GKPYVV (SEQ IDNO:192), GKPWIL (SEQ ID NO:193), GKPWII (SEQ ID NO:194), GKPWIV (SEQ IDNO:195), GKPWLL (SEQ ID NO:196), GKPWLI (SEQ ID NO:197), GKPWLV (SEQ IDNO:198), GKPWVL (SEQ ID NO:199), GKPWVI (SEQ ID NO:200), GKPWVV (SEQ IDNO:201), GRPYIL (SEQ ID NO:202), GRPYII (SEQ ID NO:203), GRPYIV (SEQ IDNO:204), GRPYLL (SEQ ID NO:205), GRPYLI (SEQ ID NO:206), GRPYLV (SEQ IDNO:207), GRPYVL (SEQ ID NO:208), GRPYVI (SEQ ID NO:209), GRPYVV (SEQ IDNO:210), GRPWIL (SEQ ID NO:211), GRPWII (SEQ ID NO:212), GRPWIV (SEQ IDNO:213), GRPWLL (SEQ ID NO:214), GRPWLI (SEQ ID NO:215), GRPWLV (SEQ IDNO:216), GRPWVL (SEQ ID NO:217), GRPWVI (SEQ ID NO:218), and GRPWVV (SEQID NO:219).

In some specific embodiments, the polypeptide of the invention consistsof or comprises an amino acid sequence selected from the groupconsisting of SDKPYIL (SEQ ID NO:220), SDKPYII (SEQ ID NO:221), SDKPYIV(SEQ ID NO:222), SDKPYLL (SEQ ID NO:223), SDKPYLI (SEQ ID NO:224),SDKPYLV (SEQ ID NO:225), SDKPYVL (SEQ ID NO:226), SDKPYVI (SEQ IDNO:227), SDKPYVV (SEQ ID NO:228), SDKPWIL (SEQ ID NO:229), SDKPWII (SEQID NO:230), SDKPWIV (SEQ ID NO:231), SDKPWLL (SEQ ID NO:232), SDKPWLI(SEQ ID NO:233), SDKPWLV (SEQ ID NO:234), SDKPWVL (SEQ ID NO:235),SDKPWVI (SEQ ID NO:236), SDKPWVV (SEQ ID NO:237), SDRPYIL (SEQ IDNO:238), SDRPYII (SEQ ID NO:239), SDRPYIV (SEQ ID NO:240), SDRPYLL (SEQID NO:241), SDRPYLI (SEQ ID NO:242), SDRPYLV (SEQ ID NO:243), SDRPYVL(SEQ ID NO:244), SDRPYVI (SEQ ID NO:245), SDRPYVV (SEQ ID NO:246),SDRPWIL (SEQ ID NO:247), SDRPWII (SEQ ID NO:248), SDRPWIV (SEQ IDNO:249), SDRPWLL (SEQ ID NO:250), SDRPWLI (SEQ ID NO:251), SDRPWLV (SEQID NO:252), SDRPWVL (SEQ ID NO:253), SDRPWVI (SEQ ID NO:254), SDRPWVV(SEQ ID NO:255), SEKPYIL (SEQ ID NO:256), SEKPYII (SEQ ID NO:257),SEKPYIV (SEQ ID NO:258), SEKPYLL (SEQ ID NO:259), SEKPYLI (SEQ IDNO:260), SEKPYLV (SEQ ID NO:261), SEKPYVL (SEQ ID NO:262), SEKPYVI (SEQID NO:263), SEKPYVV (SEQ ID NO:264), SEKPWIL (SEQ ID NO:265), SEKPWII(SEQ ID NO:266), SEKPWIV (SEQ ID NO:267), SEKPWLL (SEQ ID NO:268),SEKPWLI (SEQ ID NO:269), SEKPWLV (SEQ ID NO:270), SEKPWVL (SEQ IDNO:271), SEKPWVI (SEQ ID NO:272), SEKPWVV (SEQ ID NO:273), SERPYIL (SEQID NO:274), SERPYII (SEQ ID NO:275), SERPYIV (SEQ ID NO:276), SERPYLL(SEQ ID NO:277), SERPYLI (SEQ ID NO:278), SERPYLV (SEQ ID NO:279),SERPYVL (SEQ ID NO:280), SERPYVI (SEQ ID NO:281), SERPYVV (SEQ IDNO:282), SERPWIL (SEQ ID NO:283), SERPWII (SEQ ID NO:284), SERPWIV (SEQID NO:285), SERPWLL (SEQ ID NO:286), SERPWLI (SEQ ID NO:287), SERPWLV(SEQ ID NO:288), SERPWVL (SEQ ID NO:289), SERPWVI (SEQ ID NO:290),SERPWVV (SEQ ID NO:291), TDKPYIL (SEQ ID NO:292), TDKPYII (SEQ IDNO:293), TDKPYIV (SEQ ID NO:294), TDKPYLL (SEQ ID NO:295), TDKPYLI (SEQID NO:296), TDKPYLV (SEQ ID NO:297), TDKPYVL (SEQ ID NO:298), TDKPYVI(SEQ ID NO:299), TDKPYVV (SEQ ID NO:300), TDKPWIL (SEQ ID NO:301),TDKPWII (SEQ ID NO:302), TDKPWIV (SEQ ID NO:303), TDKPWLL (SEQ IDNO:304), TDKPWLI (SEQ ID NO:305), TDKPWLV (SEQ ID NO:306), TDKPWVL (SEQID NO:307), TDKPWVI (SEQ ID NO:308), TDKPWVV (SEQ ID NO:309), TDRPYIL(SEQ ID NO:310), TDRPYII (SEQ ID NO:311), TDRPYIV (SEQ ID NO:312),TDRPYLL (SEQ ID NO:313), TDRPYLI (SEQ ID NO:314), TDRPYLV (SEQ IDNO:315), TDRPYVL (SEQ ID NO:316), TDRPYVI (SEQ ID NO:317), TDRPYVV (SEQID NO:318), TDRPWIL (SEQ ID NO:319), TDRPWII (SEQ ID NO:320), TDRPWIV(SEQ ID NO:321), TDRPWLL (SEQ ID NO:322), TDRPWLI (SEQ ID NO:323),TDRPWLV (SEQ ID NO:324), TDRPWVL (SEQ ID NO:325), TDRPWVI (SEQ IDNO:326), TDRPWVV (SEQ ID NO:327), TEKPYIL (SEQ ID NO:328), TEKPYII (SEQID NO:329), TEKPYIV (SEQ ID NO:330), TEKPYLL (SEQ ID NO:331), TEKPYLI(SEQ ID NO:332), TEKPYLV (SEQ ID NO:333), TEKPYVL (SEQ ID NO:334),TEKPYVI (SEQ ID NO:335), TEKPYVV (SEQ ID NO:336), TEKPWIL (SEQ IDNO:337), TEKPWII (SEQ ID NO:338), TEKPWIV (SEQ ID NO:339), TEKPWLL (SEQID NO:340), TEKPWLI (SEQ ID NO:341), TEKPWLV (SEQ ID NO:342), TEKPWVL(SEQ ID NO:343), TEKPWVI (SEQ ID NO:344), TEKPWVV (SEQ ID NO:345),TERPYIL (SEQ ID NO:346), TERPYII (SEQ ID NO:347), TERPYIV (SEQ IDNO:348), TERPYLL (SEQ ID NO:349), TERPYLI (SEQ ID NO:350), TERPYLV (SEQID NO:351), TERPYVL (SEQ ID NO:352), TERPYVI (SEQ ID NO:353), TERPYVV(SEQ ID NO:354), TERPWIL (SEQ ID NO:355), TERPWII (SEQ ID NO:356),TERPWIV (SEQ ID NO:357), TERPWLL (SEQ ID NO:358), TERPWLI (SEQ IDNO:359), TERPWLV (SEQ ID NO:360), TERPWVL (SEQ ID NO:361), TERPWVI (SEQID NO:362), and TERPWVV (SEQ ID NO:363).

In some specific embodiments, the polypeptide of the invention consistsof or comprises an amino acid sequence selected from the groupconsisting of ASDKPYII (SEQ ID NO:364), ASDKPYIV (SEQ ID NO:365),ASDKPYLL (SEQ ID NO:366), ASDKPYLI (SEQ ID NO:367), ASDKPYLV (SEQ IDNO:368), ASDKPYVL (SEQ ID NO:369), ASDKPYVI (SEQ ID NO:370), ASDKPYVV(SEQ ID NO:371), ASDKPWIL (SEQ ID NO:372), ASDKPWII (SEQ ID NO:373),ASDKPWIV (SEQ ID NO:374), ASDKPWLL (SEQ ID NO:375), ASDKPWLI (SEQ IDNO:376), ASDKPWLV (SEQ ID NO:377), ASDKPWVL (SEQ ID NO:378), ASDKPWVI(SEQ ID NO:379), ASDKPWVV (SEQ ID NO:380), ASDRPYIL (SEQ ID NO:381),ASDRPYII (SEQ ID NO:382), ASDRPYIV (SEQ ID NO:383), ASDRPYLL (SEQ IDNO:384), ASDRPYLI (SEQ ID NO:385), ASDRPYLV (SEQ ID NO:386), ASDRPYVL(SEQ ID NO:387), ASDRPYVI (SEQ ID NO:388), ASDRPYVV (SEQ ID NO:389),ASDRPWIL (SEQ ID NO:390), ASDRPWII (SEQ ID NO:391), ASDRPWIV (SEQ IDNO:392), ASDRPWLL (SEQ ID NO:393), ASDRPWLI (SEQ ID NO:394), ASDRPWLV(SEQ ID NO:395), ASDRPWVL (SEQ ID NO:396), ASDRPWVI (SEQ ID NO:397),ASDRPWVV (SEQ ID NO:398), ASEKPYIL (SEQ ID NO:399), ASEKPYII (SEQ IDNO:400), ASEKPYIV (SEQ ID NO:401), ASEKPYLL (SEQ ID NO:402), ASEKPYLI(SEQ ID NO:403), ASEKPYLV (SEQ ID NO:404), ASEKPYVL (SEQ ID NO:405),ASEKPYVI (SEQ ID NO:406), ASEKPYVV (SEQ ID NO:407), ASEKPWIL (SEQ IDNO:408), ASEKPWII (SEQ ID NO:409), ASEKPWIV (SEQ ID NO:410), ASEKPWLL(SEQ ID NO:411), ASEKPWLI (SEQ ID NO:412), ASEKPWLV (SEQ ID NO:413),ASEKPWVL (SEQ ID NO:414), ASEKPWVI (SEQ ID NO:415), ASEKPWVV (SEQ IDNO:416), ASERPYIL (SEQ ID NO:417), ASERPYII (SEQ ID NO:418), ASERPYIV(SEQ ID NO:419), ASERPYLL (SEQ ID NO:420), ASERPYLI (SEQ ID NO:421),ASERPYLV (SEQ ID NO:422), ASERPYVL (SEQ ID NO:423), ASERPYVI (SEQ IDNO:424), ASERPYVV (SEQ ID NO:425), ASERPWIL (SEQ ID NO:426), ASERPWII(SEQ ID NO:427), ASERPWIV (SEQ ID NO:428), ASERPWLL (SEQ ID NO:429),ASERPWLI (SEQ ID NO:430), ASERPWLV (SEQ ID NO:431), ASERPWVL (SEQ IDNO:432), ASERPWVI (SEQ ID NO:433), ASERPWVV (SEQ ID NO:434), ATDKPYIL(SEQ ID NO:435), ATDKPYII (SEQ ID NO:436), ATDKPYIV (SEQ ID NO:437),ATDKPYLL (SEQ ID NO:438), ATDKPYLI (SEQ ID NO:439), ATDKPYLV (SEQ IDNO:440), ATDKPYVL (SEQ ID NO:441), ATDKPYVI (SEQ ID NO:442), ATDKPYVV(SEQ ID NO:443), ATDKPWIL (SEQ ID NO:444), ATDKPWII (SEQ ID NO:445),ATDKPWIV (SEQ ID NO:446), ATDKPWLL (SEQ ID NO:447), ATDKPWLI (SEQ IDNO:448), ATDKPWLV (SEQ ID NO:449), ATDKPWVL (SEQ ID NO:450), ATDKPWVI(SEQ ID NO:451), ATDKPWVV (SEQ ID NO:452), ATDRPYIL (SEQ ID NO:453),ATDRPYII (SEQ ID NO:454), ATDRPYIV (SEQ ID NO:455), ATDRPYLL (SEQ IDNO:456), ATDRPYLI (SEQ ID NO:457), ATDRPYLV (SEQ ID NO:458), ATDRPYVL(SEQ ID NO:459), ATDRPYVI (SEQ ID NO:460), ATDRPYVV (SEQ ID NO:461),ATDRPWIL (SEQ ID NO:462), ATDRPWII (SEQ ID NO:463), ATDRPWIV (SEQ IDNO:464), ATDRPWLL (SEQ ID NO:465), ATDRPWLI (SEQ ID NO:466), ATDRPWLV(SEQ ID NO:467), ATDRPWVL (SEQ ID NO:468), ATDRPWVI (SEQ ID NO:469),ATDRPWVV (SEQ ID NO:470), ATEKPYIL (SEQ ID NO:471), ATEKPYII (SEQ IDNO:472), ATEKPYIV (SEQ ID NO:473), ATEKPYLL (SEQ ID NO:474), ATEKPYLI(SEQ ID NO:475), ATEKPYLV (SEQ ID NO:476), ATEKPYVL (SEQ ID NO:477),ATEKPYVI (SEQ ID NO:478), ATEKPYVV (SEQ ID NO:479), ATEKPWIL (SEQ IDNO:480), ATEKPWII (SEQ ID NO:481), ATEKPWIV (SEQ ID NO:482), ATEKPWLL(SEQ ID NO:483), ATEKPWLI (SEQ ID NO:484), ATEKPWLV (SEQ ID NO:485),ATEKPWVL (SEQ ID NO:486), ATEKPWVI (SEQ ID NO:487), ATEKPWVV (SEQ IDNO:488), ATERPYIL (SEQ ID NO:489), ATERPYII (SEQ ID NO:490), ATERPYIV(SEQ ID NO:491), ATERPYLL (SEQ ID NO:492), ATERPYLI (SEQ ID NO:493),ATERPYLV (SEQ ID NO:494), ATERPYVL (SEQ ID NO:495), ATERPYVI (SEQ IDNO:496), ATERPYVV (SEQ ID NO:497), ATERPWIL (SEQ ID NO:498), ATERPWII(SEQ ID NO:499), ATERPWIV (SEQ ID NO:500), ATERPWLL (SEQ ID NO:501),ATERPWLI (SEQ ID NO:502), ATERPWLV (SEQ ID NO:503), ATERPWVL (SEQ IDNO:504), ATERPWVI (SEQ ID NO:505), ATERPWVV (SEQ ID NO:506), LSDKPYIL(SEQ ID NO:507), LSDKPYII (SEQ ID NO:508), LSDKPYIV (SEQ ID NO:509),LSDKPYLL (SEQ ID NO:510), LSDKPYLI (SEQ ID NO:511), LSDKPYLV (SEQ IDNO:512), LSDKPYVL (SEQ ID NO:513), LSDKPYVI (SEQ ID NO:514), LSDKPYVV(SEQ ID NO:515), LSDKPWIL (SEQ ID NO:516), LSDKPWII (SEQ ID NO:517),LSDKPWIV (SEQ ID NO:518), LSDKPWLL (SEQ ID NO:519), LSDKPWLI (SEQ IDNO:520), LSDKPWLV (SEQ ID NO:521), LSDKPWVL (SEQ ID NO:522), LSDKPWVI(SEQ ID NO:523), LSDKPWVV (SEQ ID NO:524), LSDRPYIL (SEQ ID NO:525),LSDRPYII (SEQ ID NO:526), LSDRPYIV (SEQ ID NO:527), LSDRPYLL (SEQ IDNO:528), LSDRPYLI (SEQ ID NO:529), LSDRPYLV (SEQ ID NO:530), LSDRPYVL(SEQ ID NO:531), LSDRPYVI (SEQ ID NO:532), LSDRPYVV (SEQ ID NO:533),LSDRPWIL (SEQ ID NO:534), LSDRPWII (SEQ ID NO:535), LSDRPWIV (SEQ IDNO:536), LSDRPWLL (SEQ ID NO:537), LSDRPWLI (SEQ ID NO:538), LSDRPWLV(SEQ ID NO:539), LSDRPWVL (SEQ ID NO:540), LSDRPWVI (SEQ ID NO:541),LSDRPWVV (SEQ ID NO:542), LSEKPYIL (SEQ ID NO:543), LSEKPYII (SEQ IDNO:544), LSEKPYIV (SEQ ID NO:545), LSEKPYLL (SEQ ID NO:546), LSEKPYLI(SEQ ID NO:547), LSEKPYLV (SEQ ID NO:548), LSEKPYVL (SEQ ID NO:549),LSEKPYVI (SEQ ID NO:550), LSEKPYVV (SEQ ID NO:551), LSEKPWIL (SEQ IDNO:552), LSEKPWII (SEQ ID NO:553), LSEKPWIV (SEQ ID NO:554), LSEKPWLL(SEQ ID NO:555), LSEKPWLI (SEQ ID NO:556), LSEKPWLV (SEQ ID NO:557),LSEKPWVL (SEQ ID NO:558), LSEKPWVI (SEQ ID NO:559), LSEKPWVV (SEQ IDNO:560), LSERPYIL (SEQ ID NO:561), LSERPYII (SEQ ID NO:562), LSERPYIV(SEQ ID NO:563), LSERPYLL (SEQ ID NO:564), LSERPYLI (SEQ ID NO:565),LSERPYLV (SEQ ID NO:566), LSERPYVL (SEQ ID NO:567), LSERPYVI (SEQ IDNO:568), LSERPYVV (SEQ ID NO:569), LSERPWIL (SEQ ID NO:570), LSERPWII(SEQ ID NO:571), LSERPWIV (SEQ ID NO:572), LSERPWLL (SEQ ID NO:573),LSERPWLI (SEQ ID NO:574), LSERPWLV (SEQ ID NO:575), LSERPWVL (SEQ IDNO:576), LSERPWVI (SEQ ID NO:577), LSERPWVV (SEQ ID NO:578), LTDKPYIL(SEQ ID NO:579), LTDKPYII (SEQ ID NO:580), LTDKPYIV (SEQ ID NO:581),LTDKPYLL (SEQ ID NO:582), LTDKPYLI (SEQ ID NO:583), LTDKPYLV (SEQ IDNO:584), LTDKPYVL (SEQ ID NO:585), LTDKPYVI (SEQ ID NO:586), LTDKPYVV(SEQ ID NO:587), LTDKPWIL (SEQ ID NO:588), LTDKPWII (SEQ ID NO:589),LTDKPWIV (SEQ ID NO:590), LTDKPWLL (SEQ ID NO:591), LTDKPWLI (SEQ IDNO:592), LTDKPWLV (SEQ ID NO:593), LTDKPWVL (SEQ ID NO:594), LTDKPWVI(SEQ ID NO:595), LTDKPWVV (SEQ ID NO:596), LTDRPYIL (SEQ ID NO:597),LTDRPYII (SEQ ID NO:598), LTDRPYIV (SEQ ID NO:599), LTDRPYLL (SEQ IDNO:600), LTDRPYLI (SEQ ID NO:601), LTDRPYLV (SEQ ID NO:602), LTDRPYVL(SEQ ID NO:603), LTDRPYVI (SEQ ID NO:604), LTDRPYVV (SEQ ID NO:605),LTDRPWIL (SEQ ID NO:606), LTDRPWII (SEQ ID NO:607), LTDRPWIV (SEQ IDNO:608), LTDRPWLL (SEQ ID NO:609), LTDRPWLI (SEQ ID NO:610), LTDRPWLV(SEQ ID NO:611), LTDRPWVL (SEQ ID NO:612), LTDRPWVI (SEQ ID NO:613),LTDRPWVV (SEQ ID NO:614), LTEKPYIL (SEQ ID NO:615), LTEKPYII (SEQ IDNO:616), LTEKPYIV (SEQ ID NO:617), LTEKPYLL (SEQ ID NO:618), LTEKPYLI(SEQ ID NO:619), LTEKPYLV (SEQ ID NO:620), LTEKPYVL (SEQ ID NO:621),LTEKPYVI (SEQ ID NO:622), LTEKPYVV (SEQ ID NO:623), LTEKPWIL (SEQ IDNO:624), LTEKPWII (SEQ ID NO:625), LTEKPWIV (SEQ ID NO:626), LTEKPWLL(SEQ ID NO:627), LTEKPWLI (SEQ ID NO:628), LTEKPWLV (SEQ ID NO:629),LTEKPWVL (SEQ ID NO:630), LTEKPWVI (SEQ ID NO:631), LTEKPWVV (SEQ IDNO:632), LTERPYIL (SEQ ID NO:633), LTERPYII (SEQ ID NO:634), LTERPYIV(SEQ ID NO:635), LTERPYLL (SEQ ID NO:636), LTERPYLI (SEQ ID NO:637),LTERPYLV (SEQ ID NO:638), LTERPYVL (SEQ ID NO:639), LTERPYVI (SEQ IDNO:640), LTERPYVV (SEQ ID NO:641), LTERPWIL (SEQ ID NO:642), LTERPWII(SEQ ID NO:643), LTERPWIV (SEQ ID NO:644), LTERPWLL (SEQ ID NO:645),LTERPWLI (SEQ ID NO:646), LTERPWLV (SEQ ID NO:647), LTERPWVL (SEQ IDNO:648), LTERPWVI (SEQ ID NO:649), LTERPWVV (SEQ ID NO:650), ISDKPYIL(SEQ ID NO:651), ISDKPYII (SEQ ID NO:652), ISDKPYIV (SEQ ID NO:653),ISDKPYLL (SEQ ID NO:654), ISDKPYLI (SEQ ID NO:655), ISDKPYLV (SEQ IDNO:656), ISDKPYVL (SEQ ID NO:657), ISDKPYVI (SEQ ID NO:658), ISDKPYVV(SEQ ID NO:659), ISDKPWIL (SEQ ID NO:660), ISDKPWII (SEQ ID NO:661),ISDKPWIV (SEQ ID NO:662), ISDKPWLL (SEQ ID NO:663), ISDKPWLI (SEQ IDNO:664), ISDKPWLV (SEQ ID NO:665), ISDKPWVL (SEQ ID NO:666), ISDKPWVI(SEQ ID NO:667), ISDKPWVV (SEQ ID NO:668), ISDRPYIL (SEQ ID NO:669),ISDRPYII (SEQ ID NO:670), ISDRPYIV (SEQ ID NO:671), ISDRPYLL (SEQ IDNO:672), ISDRPYLI (SEQ ID NO:673), ISDRPYLV (SEQ ID NO:674), ISDRPYVL(SEQ ID NO:675), ISDRPYVI (SEQ ID NO:676), ISDRPYVV (SEQ ID NO:677),ISDRPWIL (SEQ ID NO:678), ISDRPWII (SEQ ID NO:679), ISDRPWIV (SEQ IDNO:680), ISDRPWLL (SEQ ID NO:681), ISDRPWLI (SEQ ID NO:682), ISDRPWLV(SEQ ID NO:683), ISDRPWVL (SEQ ID NO:684), ISDRPWVI (SEQ ID NO:685),ISDRPWVV (SEQ ID NO:686), ISEKPYIL (SEQ ID NO:687), ISEKPYII (SEQ IDNO:688), ISEKPYIV (SEQ ID NO:689), ISEKPYLL (SEQ ID NO:690), ISEKPYLI(SEQ ID NO:691), ISEKPYLV (SEQ ID NO:692), ISEKPYVL (SEQ ID NO:693),ISEKPYVI (SEQ ID NO:694), ISEKPYVV (SEQ ID NO:695), ISEKPWIL (SEQ IDNO:696), ISEKPWII (SEQ ID NO:697), ISEKPWIV (SEQ ID NO:698), ISEKPWLL(SEQ ID NO:699), ISEKPWLI (SEQ ID NO:700), ISEKPWLV (SEQ ID NO:701),ISEKPWVL (SEQ ID NO:702), ISEKPWVI (SEQ ID NO:703), ISEKPWVV (SEQ IDNO:704), ISERPYIL (SEQ ID NO:705), ISERPYII (SEQ ID NO:706), ISERPYIV(SEQ ID NO:707), ISERPYLL (SEQ ID NO:708), ISERPYLI (SEQ ID NO:709),ISERPYLV (SEQ ID NO:710), ISERPYVL (SEQ ID NO:711), ISERPYVI (SEQ IDNO:712), ISERPYVV (SEQ ID NO:713), ISERPWIL (SEQ ID NO:714), ISERPWII(SEQ ID NO:715), ISERPWIV (SEQ ID NO:716), ISERPWLL (SEQ ID NO:717),ISERPWLI (SEQ ID NO:718), ISERPWLV (SEQ ID NO:719), ISERPWVL (SEQ IDNO:720), ISERPWVI (SEQ ID NO:721), ISERPWVV (SEQ ID NO:722), ITDKPYIL(SEQ ID NO:723), ITDKPYII (SEQ ID NO:724), ITDKPYIV (SEQ ID NO:725),ITDKPYLL (SEQ ID NO:726), ITDKPYLI (SEQ ID NO:727), ITDKPYLV (SEQ IDNO:728), ITDKPYVL (SEQ ID NO:729), ITDKPYVI (SEQ ID NO:730), ITDKPYVV(SEQ ID NO:731), ITDKPWIL (SEQ ID NO:732), ITDKPWII (SEQ ID NO:733),ITDKPWIV (SEQ ID NO:734), ITDKPWLL (SEQ ID NO:735), ITDKPWLI (SEQ IDNO:736), ITDKPWLV (SEQ ID NO:737), ITDKPWVL (SEQ ID NO:738), ITDKPWVI(SEQ ID NO:739), ITDKPWVV (SEQ ID NO:740), ITDRPYIL (SEQ ID NO:741),ITDRPYII (SEQ ID NO:742), ITDRPYIV (SEQ ID NO:743), ITDRPYLL (SEQ IDNO:744), ITDRPYLI (SEQ ID NO:745), ITDRPYLV (SEQ ID NO:746), ITDRPYVL(SEQ ID NO:747), ITDRPYVI (SEQ ID NO:748), ITDRPYVV (SEQ ID NO:749),ITDRPWIL (SEQ ID NO:750), ITDRPWII (SEQ ID NO:751), ITDRPWIV (SEQ IDNO:752), ITDRPWLL (SEQ ID NO:753), ITDRPWLI (SEQ ID NO:754), ITDRPWLV(SEQ ID NO:755), ITDRPWVL (SEQ ID NO:756), ITDRPWVI (SEQ ID NO:757),ITDRPWVV (SEQ ID NO:758), ITEKPYIL (SEQ ID NO:759), ITEKPYII (SEQ IDNO:760), ITEKPYIV (SEQ ID NO:761), ITEKPYLL (SEQ ID NO:762), ITEKPYLI(SEQ ID NO:763), ITEKPYLV (SEQ ID NO:764), ITEKPYVL (SEQ ID NO:765),ITEKPYVI (SEQ ID NO:766), ITEKPYVV (SEQ ID NO:767), ITEKPWIL (SEQ IDNO:768), ITEKPWII (SEQ ID NO:769), ITEKPWIV (SEQ ID NO:770), ITEKPWLL(SEQ ID NO:771), ITEKPWLI (SEQ ID NO:772), ITEKPWLV (SEQ ID NO:773),ITEKPWVL (SEQ ID NO:774), ITEKPWVI (SEQ ID NO:775), ITEKPWVV (SEQ IDNO:776), ITERPYIL (SEQ ID NO:777), ITERPYII (SEQ ID NO:778), ITERPYIV(SEQ ID NO:779), ITERPYLL (SEQ ID NO:780), ITERPYLI (SEQ ID NO:781),ITERPYLV (SEQ ID NO:782), ITERPYVL (SEQ ID NO:783), ITERPYVI (SEQ IDNO:784), ITERPYVV (SEQ ID NO:785), ITERPWIL (SEQ ID NO:786), ITERPWII(SEQ ID NO:787), ITERPWIV (SEQ ID NO:788), ITERPWLL (SEQ ID NO:789),ITERPWLI (SEQ ID NO:790), ITERPWLV (SEQ ID NO:791), ITERPWVL (SEQ IDNO:792), ITERPWVI (SEQ ID NO:793), ITERPWVV (SEQ ID NO:794), VSDKPYIL(SEQ ID NO:795), VSDKPYII (SEQ ID NO:796), VSDKPYIV (SEQ ID NO:797),VSDKPYLL (SEQ ID NO:798), VSDKPYLI (SEQ ID NO:799), VSDKPYLV (SEQ IDNO:800), VSDKPYVL (SEQ ID NO:801), VSDKPYVI (SEQ ID NO:802), VSDKPYVV(SEQ ID NO:803), VSDKPWIL (SEQ ID NO:804), VSDKPWII (SEQ ID NO:805),VSDKPWIV (SEQ ID NO:806), VSDKPWLL (SEQ ID NO:807), VSDKPWLI (SEQ IDNO:808), VSDKPWLV (SEQ ID NO:809), VSDKPWVL (SEQ ID NO:810), VSDKPWVI(SEQ ID NO:811), VSDKPWVV (SEQ ID NO:812), VSDRPYIL (SEQ ID NO:813),VSDRPYII (SEQ ID NO:814), VSDRPYIV (SEQ ID NO:815), VSDRPYLL (SEQ IDNO:816), VSDRPYLI (SEQ ID NO:817), VSDRPYLV (SEQ ID NO:818), VSDRPYVL(SEQ ID NO:819), VSDRPYVI (SEQ ID NO:820), VSDRPYVV (SEQ ID NO:821),VSDRPWIL (SEQ ID NO:822), VSDRPWII (SEQ ID NO:823), VSDRPWIV (SEQ IDNO:824), VSDRPWLL (SEQ ID NO:825), VSDRPWLI (SEQ ID NO:826), VSDRPWLV(SEQ ID NO:827), VSDRPWVL (SEQ ID NO:828), VSDRPWVI (SEQ ID NO:829),VSDRPWVV (SEQ ID NO:830), VSEKPYIL (SEQ ID NO:831), VSEKPYII (SEQ IDNO:832), VSEKPYIV (SEQ ID NO:833), VSEKPYLL (SEQ ID NO:834), VSEKPYLI(SEQ ID NO:835), VSEKPYLV (SEQ ID NO:836), VSEKPYVL (SEQ ID NO:837),VSEKPYVI (SEQ ID NO:838), VSEKPYVV (SEQ ID NO:839), VSEKPWIL (SEQ IDNO:840), VSEKPWII (SEQ ID NO:841), VSEKPWIV (SEQ ID NO:842), VSEKPWLL(SEQ ID NO:843), VSEKPWLI (SEQ ID NO:844), VSEKPWLV (SEQ ID NO:845),VSEKPWVL (SEQ ID NO:846), VSEKPWVI (SEQ ID NO:847), VSEKPWVV (SEQ IDNO:848), VSERPYIL (SEQ ID NO:849), VSERPYII (SEQ ID NO:850), VSERPYIV(SEQ ID NO:851), VSERPYLL (SEQ ID NO:852), VSERPYLI (SEQ ID NO:853),VSERPYLV (SEQ ID NO:854), VSERPYVL (SEQ ID NO:855), VSERPYVI (SEQ IDNO:856), VSERPYVV (SEQ ID NO:857), VSERPWIL (SEQ ID NO:858), VSERPWII(SEQ ID NO:859), VSERPWIV (SEQ ID NO:860), VSERPWLL (SEQ ID NO:861),VSERPWLI (SEQ ID NO:862), VSERPWLV (SEQ ID NO:863), VSERPWVL (SEQ IDNO:864), VSERPWVI (SEQ ID NO:865), VSERPWVV (SEQ ID NO:866), VTDKPYIL(SEQ ID NO:867), VTDKPYII (SEQ ID NO:868), VTDKPYIV (SEQ ID NO:869),VTDKPYLL (SEQ ID NO:870), VTDKPYLI (SEQ ID NO:871), VTDKPYLV (SEQ IDNO:872), VTDKPYVL (SEQ ID NO:873), VTDKPYVI (SEQ ID NO:874), VTDKPYVV(SEQ ID NO:875), VTDKPWIL (SEQ ID NO:876), VTDKPWII (SEQ ID NO:877),VTDKPWIV (SEQ ID NO:878), VTDKPWLL (SEQ ID NO:879), VTDKPWLI (SEQ IDNO:880), VTDKPWLV (SEQ ID NO:881), VTDKPWVL (SEQ ID NO:882), VTDKPWVI(SEQ ID NO:883), VTDKPWVV (SEQ ID NO:884), VTDRPYIL (SEQ ID NO:885),VTDRPYII (SEQ ID NO:886), VTDRPYIV (SEQ ID NO:887), VTDRPYLL (SEQ IDNO:888), VTDRPYLI (SEQ ID NO:889), VTDRPYLV (SEQ ID NO:890), VTDRPYVL(SEQ ID NO:891), VTDRPYVI (SEQ ID NO:892), VTDRPYVV (SEQ ID NO:893),VTDRPWIL (SEQ ID NO:894), VTDRPWII (SEQ ID NO:895), VTDRPWIV (SEQ IDNO:896), VTDRPWLL (SEQ ID NO:897), VTDRPWLI (SEQ ID NO:898), VTDRPWLV(SEQ ID NO:899), VTDRPWVL (SEQ ID NO:900), VTDRPWVI (SEQ ID NO:901),VTDRPWVV (SEQ ID NO:902), VTEKPYIL (SEQ ID NO:903), VTEKPYII (SEQ IDNO:904), VTEKPYIV (SEQ ID NO:905), VTEKPYLL (SEQ ID NO:906), VTEKPYLI(SEQ ID NO:907), VTEKPYLV (SEQ ID NO:908), VTEKPYVL (SEQ ID NO:909),VTEKPYVI (SEQ ID NO:910), VTEKPYVV (SEQ ID NO:911), VTEKPWIL (SEQ IDNO:912), VTEKPWII (SEQ ID NO:913), VTEKPWIV (SEQ ID NO:914), VTEKPWLL(SEQ ID NO:915), VTEKPWLI (SEQ ID NO:916), VTEKPWLV (SEQ ID NO:917),VTEKPWVL (SEQ ID NO:918), VTEKPWVI (SEQ ID NO:919), VTEKPWVV (SEQ IDNO:920), VTERPYIL (SEQ ID NO:921), VTERPYII (SEQ ID NO:922), VTERPYIV(SEQ ID NO:923), VTERPYLL (SEQ ID NO:924), VTERPYLI (SEQ ID NO:925),VTERPYLV (SEQ ID NO:926), VTERPYVL (SEQ ID NO:927), VTERPYVI (SEQ IDNO:928), VTERPYVV (SEQ ID NO:929), VTERPWIL (SEQ ID NO:930), VTERPWII(SEQ ID NO:931), VTERPWIV (SEQ ID NO:932), VTERPWLL (SEQ ID NO:933),VTERPWLI (SEQ ID NO:934), VTERPWLV (SEQ ID NO:935), VTERPWVL (SEQ IDNO:936), VTERPWVI (SEQ ID NO:937), and VTERPWVV (SEQ ID NO:938).

In some specific embodiments, the polypeptide of the invention consistsof or comprises an amino acid sequence derived from Alpha-actinin-1,such as a sequence selected from ASDKPYIL (SEQ ID NO:6), AGDKNYIL (SEQID NO:10), AGDKNYIT (SEQ ID NO:11), AGDKSYIT (SEQ ID NO:12), ADGKPYIV(SEQ ID NO:13), and AEDKDFIT (SEQ ID NO:14).

In some specific embodiments, the polypeptide of the invention consistsof or comprises an amino acid sequence derived from Alpha-actinin-2,such as a sequence selected from ASDKPYIL (SEQ ID NO:6), AADKPYIL (SEQID NO:15), AGDKNYIT (SEQ ID NO:11), ATDKPYIL (SEQ ID NO:16), AGDKPYIT(SEQ ID NO:17), ASEKPYIL (SEQ ID NO:18), ADGKPYVT (SEQ ID NO:19),AGDKPYIL (SEQ ID NO:20), ASDKPNIL (SEQ ID NO:21), ASDKPYIT (SEQ IDNO:22), AADKPFIL (SEQ ID NO:23), ASDKAYIT (SEQ ID NO:24), AGDKAYIT (SEQID NO:25), ANGKPFIT (SEQ ID NO:26), and AGDKNFIT (SEQ ID NO:27).

In some specific embodiments, the polypeptide of the invention consistsof or comprises an amino acid sequence derived from Alpha-actinin-3,such as a sequence selected from ASDKPYIL (SEQ ID NO:6), AADKPYIL (SEQID NO:15), ASDKAYIT (SEQ ID NO:24), ASDKSYIT (SEQ ID NO:28), ASDKTYIT(SEQ ID NO:29), ASDKNYIT (SEQ ID NO:30), AGDKNYIL (SEQ ID NO:10),AGDKSYIT (SEQ ID NO:12), AGDKNYIT (SEQ ID NO:11), AGDKKYIT (SEQ IDNO:31), and AGDKNYIS (SEQ ID NO:32).

In some specific embodiments, the polypeptide of the invention consistsof or comprises an amino acid sequence derived from Alpha-actinin-4,such as a sequence selected from ASDKPYIL (SEQ ID NO:6) , AGDKPYIL (SEQID NO:20), AADKNYIT (SEQ ID NO:33), AGDKNYIM (SEQ ID NO:34), AGDKNYIT(SEQ ID NO:11), AADKNFIM (SEQ ID NO:35), AADKNFIT (SEQ ID NO:36),AGDKGIRS (SEQ ID NO:37), and AGDKNFIT (SEQ ID NO:27).

The present invention further relates to compositions comprising thepolypeptides of the invention. In some embodiments the compositions ofthe invention is capable of promoting satiety in a subject uponconsumption.

In some embodiments in the compositions of the invention the amount ofsaid polypeptide in the composition is less than about 10 g, such asless than 9 g, 8 g, 7 g, 6 g, 5 g, 4 g, 3 g, 2 g, 1 g, 900 mg, 800 mg,700 mg, 600 mg, 500 mg, 400 mg, 300 mg, 200 mg, 150 mg, 100 mg, 90 mg,80 mg, 70 mg, 60 mg, 50 mg, 40 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, or5 mg.

In some embodiments in the compositions of the invention the amount ofsaid polypeptide in the composition is at least about 5 mg, such as atleast about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g,or 10 g.

In some embodiments in the compositions of the invention the energycontent derived through the process of cellular respiration is less than50 kilojoules (kJ), such as less than 40 kJ, such as less than 30 kJ,such as less than 20 kJ, such as less than 10 kJ, such as less than 5000Joules (3), such as less than 1000 J, such as less than 900 J, such asless than 800 J, such as less than 700 J, such as less than 600 J, suchas less than 500 J, such as less than 400 J, such as less than 300 J,such as less than 200 J, such as less than 100 J, such as less than 50J.

In some embodiments the compositions of the invention is a foodcomposition.

In some embodiments the compositions of the invention is a fermentedcomposition.

In some embodiments the compositions of the invention is a dairyproduct.

In some embodiments the compositions of the invention is apharmaceutical composition.

In some embodiments the compositions of the invention is a nutritionalcomposition.

In some embodiments the compositions of the invention is an oral dosageform. In some embodiments the oral dosage form is selected from thegroup comprising tablets, capsules, caplets, slurries, sachets,suspensions, chewing gum, and powder formulation that may be dissolvedin a liquid. In some embodiments the oral dosage form is a suspension.In some embodiments the oral dosage form is a powder formulation thatmay be dissolved in a liquid. In some embodiments the liquid is water,milk, juice, or yogurt.

Example 1

Assays:

Ca²⁺ Flux Assay:

Elevation of intracellular calcium level was measured using thefluorescent calcium chelating dye Fluo-4 AM (ThermoFischer Scientific,Denmark). Briefly, cells were grown as a monolayer in 96-well tissueculture plates (Sarstedt, Germany) to near confluence in appropriategrowth medium as described in the cell culture section. Prior to thestart of the assay, the cells were incubated with 1.5 μM Fluo-4 AM incomplete culture media mixed 1:1 with Hank's balanced salt solution(HBSS, ThermoFischer Scientific, Denmark) containing 25 mM HEPES (pH7.4), 1% BSA (Sigma-Aldrich, Denmark), 2% ink (Soluro GMBH, Germany),0.01% Pluronic F-127 (Sigma-Aldrich, Denmark) and 1 mM Probenecide(Sigma-Aldrich) for 60 minutes at 37° C.

All test compounds were dissolved in water, and then diluted in 1×HBSScontaining 25 mM HEPES (pH 7.4), 1% BSA and 2% ink. Without any removalof excess Fluo-4 AM, test compounds were added directly into the wellsand fluorescence were measured using instrument settings for excitationat 488 nm and emission at 525 nm in a microtiter plate reader(SpectraMax M5, Molecular Devices, USA).

Cell Culture:

Cell culture media, Dulbecco's phosphate-buffered saline, pH 7.4 (DPBS),glutamine, trypsin-EDTA and antibiotics were obtained from ThermoFischerScientific (Denmark). Fetal bovine serum and all other chemicals werepurchased from Sigma-Aldrich (Denmark), unless otherwise stated.

Murine intestinal enteroendocrine L-cell lines that expresses theproglucagon gene and secretes GLP-1 in vitro were used. Cells were grownin DMEM containing 1 g/L D-glucose, 10% fetal bovine serum, 2 mMglutamine, 1% penicillin/streptomycin/neomycin and cultured in ahumidified incubator in 95% air and 5% CO2 at 37° C.

Other murine intestinal cell lines were cultured in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 2 mM glutamine, 2.5 g/L glucose,20 mM HEPES, 60 nM sodium selenite, 5 μg/ml transferrin, 5 μg/mlinsulin, 50 nM dexamethasone, 10 nM EGF, 1 nM triiodothyronine, 2% fetalbovine serum and 1% penicillin/streptomycin/neomycin at 37° C. in 5%CO2-95% air atmosphere.

Human intestinal cell lines were cultured in McCoy's modified 5A mediumsupplemented with 10% fetal bovine serum and 1%penicillin/streptomycin/neomycin at 37° C. and 5% CO2 in a humidifiedincubator.

Cells were routinely sub-cultivated 1:3 and given new media every secondday.

Determination of GLP-1 Levels:

GLP-1 levels were determined using a sandwich enzyme-linkedimmunoabsorbant assay (ELISA). The primary antibody to GLP-1 [2.5 μg/mlmouse monoclonal (HYB 147-06) in 0.05M bicarbonate/carbonate buffer;BioPorto Diagnostics A/S, Gentofte, Denmark) was coated on a flat-bottom96-well plate (Sarstedt, Nümbrecht, Germany) for at least 24 hours at 4°C. This primary antibody is specific for the amidated C-terminus of thepeptide and reacts with GLP-1 (7-36), GLP-1 (9-36) and GLP-1 (1-36), butnot with GLP-1 (7-37). After blocking the plate using a PBS buffercontaining 4% w/v BSA (Sigma-Aldrich, Denmark) and 0.1% v/v Tween 20(Sigma-Aldrich) for 1 hour at room temperature, the plate was washedfour times with PBS buffer containing 0.1% v/v Tween 20. A standardcurve with GLP-1 peptide [human GLP-1 (7-36), Sigma-Aldrich, Denmark)concentrations ranging from 0 pg/ml to 1000 pg/ml was prepared in PBSbuffer containing 0.5% BSA and 0.05% Tween 20, and samples were dilutedif necessary. Samples and standards were added to the microtiter plateand incubated with the primary antibody for two hours at roomtemperature. Subsequently, the plate was washed four times, and thewells were incubated with a secondary biotinylated antibody to GLP-1 [1μg/ml; mouse monoclonal (ABS 033-01), BioPorto Diagnostics A/S,Gentofte, Denmark) for two hours at room temperature. After anotherwashing step, samples were incubated with streptavidin-horseradishperoxidase (1:200, Dako A/S, Denmark) for 45 minutes followed by anincubation with TMB solution (containing 3,3′,5,′5-tetramethylbenzidineand H2O2, SMS-gruppen, Denmark). The reaction was stopped by addingH2SO4 (0.2M), and the absorbance of the yellow end product was measuredat 450 nm on a microtiter plate spectrophotometer (SpectraMax M5,Molecular Devices, USA). The concentrations of the samples weredetermined by interpolation to the concentrations of the standardsolutions.

Cells (˜5×10^5 per sample) were incubated for up to 90 min in DulbeccosModified Eagle Medium (DMEM) containing 5.56 mM glucose in absence orpresence of different amounts (weight/volume) of protein hydrolysate(pig heart). Supernatant was filtered through 0.45 micron filters andassayed for content of GLP-1 as described in ELISA protocol. Data aremean+SEM from quadruplicate samples.

Preparation of Bioactive Peptides by Enzymatic Digestion of Meat

Minced meat is diluted 1-10 times with distilled water, adjusted to pH1-3 with hydrochloric acid, and incubated with 0.01-10% pepsin (w/w) at4-40° C. for ½-12 h with adequate mixing. Insoluble material is removedby centrifugation at 100-1000×g for 3-30 min, and supernatant isneutralized with NaOH. Using sterile conditions, low molecular weightpeptides in supernatant are recovered by tangential ultrafiltration at4-40° C. for ½-12 h, and excess water is evaporated at 25-50° C. for upto 12 h. The concentrated dialysate is tested for bioactivity with cellsand used for further purification by HPLC.

Purification and Identification of Bioactive Peptide ASDKPYIL (SEQ IDNO: 6)

Upconcentrated dialysates were fractionated on preparative C18 columnsusing buffer B: 20 mM phosphate buffer pH 8.25/10% ACN and a gradient of0-40% in buffer A: 60% ACN in same buffer. Fractions were tested forbioactivity and further purified by isocratic elution using EVO C18columns with 4.5% ACN in 0.1%FA isocratic for 30 min. Fractions weresubject to MS characterization, where a dominating peak with m/z 453.75(+2) was observed. Extracted ions chromatograms show this peak to bepresent in all active fractions. De novo sequencing of 453.75 peak gives[A]SDKPY[I,L][I,L]. N-terminal A (e.g., SEQ ID NO: 6) is calculated fromparent ion −A7. I and L are not resolved by MS because of equalmolecular weights. Search of protein sequences gives only ASDKPYIL(SEQID NO: 6) as match. ASDKPYIL (SEQ ID NO: 6) is only found inalpha-actinin-2, a major muscle protein.

Stability of Peptides Ex Vivo

Peptides are degraded by proteases in the gastrointestinal tract.However the speed of this degradation depends on the sequence of thepeptide. In order to measure stability of the ASDKPYIL (SEQ ID NO: 6)peptide series and to compare with e.g. RRKPYIL (SEQ ID NO: 1008), 10 or50 mg (wet weight) of mouse or rat intestinal tissue (distal ileum) wasequilibrated in V-bottom 24 well plates in 800 μl HBSS, 25 mM HEPES, pH7,4 at 37 oC with shaking at 350 rpm. Identical amounts of differentpeptides (final concentration of 1 μg/ml) were added to the intestinalpieces and incubation continued. At various time points, 100 μl aliquotswere removed and diluted into whey protein hydrolysate (finalconcentration of 10 mg/ml) to non-specifically compete proteaseactivity. Peptide solutions were then diluted and tested for bioactivity(FIG. 8). Peptides incubated under same conditions but in absence ofintestine served as controls (no degradation). Determination of EC50 forstimulation of cells allowed calculation of recovered peptide (FIG. 9),assuming simple inactivation by the tissue.

Example 2

1) Structure-Activity Relationship and Stability (SAR)

-   -   a. Extended versions    -   b. Substituted versions        2) In Vivo Studies in Mice    -   c. Acute effects on feed intake (satiety)    -   d. Long-term effects on weight may be determined

Based on structural modelling studies of DC7-2 and NTR-1 interactions,peptides being octapeptides, heptapeptides, hexapeptides, orpentapeptides to exhibit increased potency due to increased binding maybe predicted.

Comparison with SAR studies using synthetic peptides, peptides withincreased potency and stability may be both predicted and observed.

Assays

Synthetic Peptides

Based on the sequence of the natural hormone Neurotensin (QLYENKPRRPYIL,SEQ ID NO: 1009), the bioactive Neurotensin fragment NT(8-13)(RRPYIL,SEQ ID NO: 39) and the identified bioactive octapeptide DC7-2 (ASDKPYIL,SEQ ID NO: 6), synthetic peptides with systematic substitutions ofN-terminal amino acids of the octapeptide (X-SDKPYIL, SEQ ID NO: 220),the heptapeptide (X-DKPYIL, SEQ ID NO: 8), the hexapeptide (X-KPYIL, SEQID NO: 9) and the pentapeptide (X-PYIL SEQ ID NO: 4) were synthesisedusing standard techniques (Schafer-N, Denmark). All peptides weredissolved in pure HPLC-grade water and stored at −20° C.

Stability of Peptides

Concentration Determination

Protein concentration of synthetic peptides (Schafer-N, Denmark), NT(Sigma-Aldrich, Denmark) and NT (8-13)(Sigma-Aldrich, Denmark) weredetermined by measuring absorbance at 280 nm in Costar® 96-wellUV-transparent plates (Corning, Sigma-Aldrich, Denmark). Each peptidewas measured in 4 different concentrations by dilution in Hank'sbalanced salt solution (HBSS, ThermoFischer Scientific, Denmark)containing 25 mM HEPES (pH 7.4) (Sigma-Aldrich, Denmark). For stabilityassays, all peptides were diluted to 3×10⁻⁵ M in HBSS; 25 mM HEPES (Ph7.4) and stored at +4° C.

Intestine Homogenate

Small intestines from 20 Swiss-Webster males were homogenized in 350 mlDulbecco's phosphate-buffered saline (PBS) (pH 7.4) (ThermoFischerScientific, Denmark) with a IKA® basic 18 Ultra-Turrax tissuehomogenizer set a speed 5 followed by filtration using 100 μm nylon meshfilter. Protein concentration was 6 mg/ml using the bicinconinic acidassay (ThermoFischer Scientific, Denmark) and bovine serum albumin asstandard. The intestine homogenate was diluted 10 times in HBSScontaining 25 mM HEPES (pH 7.4), and further diluted 30×, 90×, 270×,810× or 2430× before incubation with peptides. All solutions wereprewarmed to 37° C. before mixing with peptide solutions.

Peptides were incubated at 10⁻⁵ M with dilutions of small intestinehomogenate at 37° C. for 90 minutes with shaking. Reactions were stoppedby addition of 1 M phosphoric acid (final 0.4 M, pH˜1.2). Each peptideincubation mix was then neutralized with NaOH to pH 7.2-7.4 andimmediately tested for activity in intestinal cells. Control for zerodegradation, i.e. addition of 1 M phosphoric acid prior to addition ofintestine homogenate, was included for each peptide.

Fetal Bovine Serum

All peptides were incubated at 10⁻⁵ M with Fetal Bovine Serum (FBS;final concentration of 66.7%) (Sigma Aldrich, Denmark) at 37° C. for 3hours. The peptide degradation was terminated using 1 M phosphoric acid(final 0.4 M, pH˜1.2) and neutralized to pH 7.2-7.4 with NaOH beforetesting activity in intestinal cells. As for small intestinehomogenates, a zero degradation control was included for each peptide.

Kinetic Studies of Selected Peptides

DC7-2, NT, DKPYIL (SEQ ID NO: 8) and NT-(8-13) (final concentration of10-6 M) were incubated either with FBS or with 270X diluted intestinalhomogenate at 37° C. for various time points with shaking. Degradationwas stopped with 1 M phosphoric acid and the samples were subsequentlyneutralized and immediately tested with intestinal cells as describedabove. Control for zero degradation was included for each peptide asabove.

Study of Hexapeptides

The 20 hexapeptides with systematic N-terminal substitutions (X-KPYIL,SEQ ID NO: 9) (Schafer-N, Denmark) and NT (8-13) was incubated at 10-6 Min either FBS for 10 minutes or with 270X diluted intestinal homogenatefor 30 minutes at 37° C. with shaking. The degradation was stopped with1 M phosphoric acid. Peptide solutions were neutralized with NaOH (pH7.2-7.4), diluted and immediately tested for bioactivity using murineintestinal cells. Determination of EC50 for stimulation of cells allowedcalculation of recovered peptide.

Systematic substitutions of N-terminal amino acids in octapeptideASDKPYIL (SEQ ID NO: 6) and their importance for activity and stability.Sequence, activity and stability of DC7-2 is indicated in grey.

Notes for Tables ¹⁾Stability in serum is expressed as fraction ofpeptide activity left after 10 min of incubation in serum at 37° C.compared with undigested sample as described in Examples. ²⁾Stability inintestine is expressed as % activity left after 30 min incubation inintestine homogenate at 37° C. as described in Examples.

Systematic substitutions of N-terminal amino acid in heptapeptideSDKPYIL(SEQ ID NO: 220) and their importance for activity and stability.Sequence, activity and stability of peptide contained in DC7-2 isindicated in grey.

Systematic substitutions of N-terminal amino acid in hexapeptide DKPYIL(SEQ ID NO: 8) and their importance for activity and stability.Sequence, activity and stability of peptide contained in DC7-2 isindicated in grey.

Systematic substitutions of N-terminal amino acid in pentapeptideKPYIL(SEQ ID NO: 9) and their importance for activity and stability.Sequence, activity and stability of peptide contained in DC7-2 isindicated in grey.

In conclusion, the results demonstrates that octa- and heptapeptides aremore stable, and that the N-terminal aa in the hexapeptide has asignificant implication on the stability.

As compared to the hexapeptide of a natural hormone, neurotensin (8-13)(NT with the sequence RRPYIL), one specific peptide of the presentinvention DKPYIL is nearly 100 times more stable in serum and around100-1000× more stable in intestine homogenate.

In Vivo Studies

Acute effects of DC7-2 on satiety is shown in FIG. 12, 17-20.

The invention claimed is:
 1. An oral dosage form comprising apolypeptide comprising at least the amino acid sequenceAA3-K-P-Y-I-L  (SEQ ID NO:1020),AA1-AA2-D-R-P-Y-I-L  (SEQ ID NO:1021),AA1-AA2-E-R-P-Y-I-L  (SEQ ID NO:1022)or AA1-AA2-G-R-P-Y-I-L  (SEQ ID NO:1023) wherein the compositioncomprises at least about 5 mg and less than about 3 g of saidpolypeptide, wherein the composition has an energy content of less than50 kilojoules (kJ), wherein AA1 is an amino acid selected from A, L, I,and V; AA2 is an amino acid selected from S, T, G, A, N, E and D; AA3 isan amino acid selected from D, E, G, Q, T, S, C, P, and N, wherein saidcomposition is an oral dosage form.
 2. The oral dosage form according toclaim 1, wherein the polypeptide comprises the sequenceAA1-AA2-AA3-K-P-Y-I-L  (SEQ ID NO:1024) orAA1-AA2-AA3-R-P-Y-I-L  (SEQ ID NO:1025); wherein AA1 is an amino acidselected from A, L, I, and V; AA2 is an amino acid selected from S, T,G, A, N, E and D; AA3 is an amino acid selected from D, E, and G.
 3. Theoral dosage form according to claim 2, wherein AA1 is A and/or whereinAA2 is S, and/or wherein AA3 is D.
 4. The oral dosage form according toclaim 1, which amino acid sequence is not found in nature.
 5. The oraldosage form according to claim 1, which amino acid sequence onlycontains natural amino acids.
 6. The oral dosage form according to claim1, wherein the polypeptide is 6-49, 6-48, 6-47, 6-46, 6-45, 6-44, 6-43,6-42, 6-41, 6-40, 6-39, 6-38, 6-37, 6-36, 6-35, 6-34, 6-33, 6-32, 6-31,6-30, 6-29, 6-28, 6-27, 6-26, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19,6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7amino acids in length, or which is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50amino acids in length.
 7. The oral dosage form according to claim 1,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of: ASDKPYIL (SEQ ID NO:6), AADKPYIL (SEQ IDNO:15), ATDKPYIL (SEQ ID NO:16), ASEKPYIL (SEQ ID NO:18), AGDKPYIL (SEQID NO:20), SDKPYIL(SEQ ID NO:7), DKPYIL (SEQ ID NO:8), EKPYIL-(SEQ IDNO:114), GKPYIL (SEQ ID NO:184), SDKPYIL (SEQ ID NO:220), SEKPYIL (SEQID NO:256), TDKPYIL (SEQ ID NO:292), TEKPYIL (SEQ ID NO:328), ASEKPYIL(SEQ ID NO:399), ATDKPYIL (SEQ ID NO:435), ATEKPYIL (SEQ ID NO:471),LSDKPYIL (SEQ ID NO:507), LSEKPYIL (SEQ ID NO:543), LTDKPYIL (SEQ IDNO:579), LTEKPYIL (SEQ ID NO:615), ISDKPYIL (SEQ ID NO:651), ISEKPYIL(SEQ ID NO:687), ITDKPYIL (SEQ ID NO:723), ITEKPYIL (SEQ ID NO:759),VSDKPYIL (SEQ ID NO:795), VSEKPYIL (SEQ ID NO:831), VTDKPYIL (SEQ IDNO:867), VTEKPYIL (SEQ ID NO:903), ASDRPYIL (SEQ ID NO:381), ASERPYIL(SEQ ID NO:417), ATDRPYIL (SEQ ID NO:453), ATERPYIL (SEQ ID NO:489),LSDRPYIL (SEQ ID NO:525), LSERPYIL (SEQ ID NO:561), LTDRPYIL (SEQ IDNO:597), LTERPYIL (SEQ ID NO:633), ISDRPYIL (SEQ ID NO:669), ISERPYIL(SEQ ID NO:705), ITDRPYIL (SEQ ID NO:741), ITERPYIL (SEQ ID NO:777),VSDRPYIL (SEQ ID NO:813), VSERPYIL (SEQ ID NO:849), VTDRPYIL (SEQ IDNO:885), and VTERPYIL (SEQ ID NO:921).
 8. The oral dosage form accordingto claim 1, wherein the amount of said polypeptide in the composition isat least about 5 mg and less than about 2 g, 1 g, 900 mg, 800 mg, 700mg, 600 mg, 500 mg, 400 mg, 300 mg, 200 mg, 150 mg, 100 mg, 90 mg, 80mg, 70 mg, 60 mg, 50 mg, 40 mg, 30 mg, 25 mg, 20 mg, 15 mg, or 10 mg. 9.The oral dosage form of claim 1, wherein the composition reduces foodintake in a subject upon consumption.
 10. The oral dosage form accordingto claim 1, wherein the polypeptide comprises the sequenceAA1-AA2-AA3-K-P-Y-I-L  (SEQ ID NO:1029) wherein AA1 is an amino acidselected from A, L, I, and V; AA2 is an amino acid selected from S, T,G, A, N, E and D; and AA3 is an amino acid selected from D, E, G, Q, T,S, C, P, and N.
 11. The oral dosage form according to claim 1, whereinthe polypeptide comprises the sequenceAA2-AA3-K-P-Y-I-L  (SEQ ID NO:1026) wherein AA2 is an amino acidselected from S, T, G, A, N, E and D; and AA3 is an amino acid selectedfrom D and E.
 12. The oral dosage form according to claim 1, wherein thepolypeptide comprises the sequenceAA1-AA2-AA3-K-P-Y-I-L  (SEQ ID NO:1027) wherein AA1 is an amino acidselected from A, L, I, and V; AA2 is an amino acid selected from S, T,G, A, N, E and D; and AA3 is an amino acid selected from D and E. 13.The oral dosage form according to claim 12, wherein AA1 is A and whereinAA2 is S, and wherein AA3 is D.
 14. The oral dosage form according toclaim 1, wherein the polypeptide consists of the sequenceAA3-K-P-Y-I-L  (SEQ ID NO:1028),AA2-AA3-K-P-Y-I-L  (SEQ ID NO:1026),or AA1-AA2-AA3-K-P-Y-I-L  (SEQ ID NO:1027) wherein AA1 is an amino acidselected from A, L, I, and V; AA2 is an amino acid selected from S, T,G, A, N, E and D; and AA3 is an amino acid selected from D and E. 15.The oral dosage form according to claim 14, wherein AA1 is A, whereinAA2 is S, and wherein AA3 is D.